CN102399870A - Reagent for determining survival and prognosis of patients with esophagus cancer - Google Patents

Abstract

The present invention provides methods and compositions for diagnosis, classification and prognosis of esophageal cancer according to the level of specific miRNAs or the corresponding gene status. The present invention also provides a composition containing components capable of reducing the level of miRNA, thereby improving the survival rate of patients with esophageal cancer.

Description

The survive reagent of prognosis of a kind of definite esophagus cancer patient

The application is that application number is 200680019815.8, the applying date is on November 28th, 2006, invention and created name is divided an application for the application for a patent for invention of " method and the compsn that are used for esophagus cancer diagnosis, prognosis and raising survival rate ".

Technical field

This application relates to the method for carrying out disease (the for example esophageal carcinoma) diagnosis, prognosis and raising survival rate according to patient's microRNA level.

Background technology

The esophageal carcinoma is gastral the third-largest cancer, also is the lethal the seventh-largest cause of global cancer.The modal type of the esophageal carcinoma is squamous cell cancer and gland cancer.Squamous cell cancer betides the pinacocyte of arranging along oesophagus.Gland cancer betides oesophagus internal layer and relevant with Barrett ' s oesophagus.

Squamous cell carcinoma of esophagus has typically follows transfer (Wen et al., Fam.Cancer.2006 May 25 from heteroplasia before attacking late period to the developmental stage of aggressive squamous cell cancer usually; [Epub ahead of print]).The esophageal carcinoma follows many expression of gene to change usually.Such as, COX-2 is relevant with heteroplasia with change hair tonic exhibition usually with VEGF, and related to cancer, relevant (Liang et al, Cancer Res.2006,66:7111-7118 with potential transfer; Vallbohmer et al, Arch Surg.2006,141:476-481; Fam.Cancer.2006 May 25, [Epub ahead of print]; Matsumoto et al, J Gastrointest Surg.2006,10:1016-1022; Soma et al, Int J Cancer.2006,119:771-782).

MicroRNA (miRNA) is the long non-coding single stranded RNAs of one type little, 22 Nucleotide, and partially or completely with the target sequence homology, interacting with the 3` non-coding region (3`-UTR) of target mRNA molecule, (Bartel, Cell 2004,116:281-297).The interaction of miRNA and target mRNA stops mRNA to be translated as albumen.In some cases, when target mRNA and miRNA accurately mated, mRNA also can degrade.MiRNA participates in a series of cell processes, for example cytodifferentiation, cell growth and necrocytosis (Cheng et al, Nucleic Acids Res 2005,33:1290-7; John et al, PLoS Biol.2004,2:e363).(Zamore and Haley, Science 2005,309:1519-1524) to have nearly 1000 miRNA according to estimates on the human genome.It possibly be that (Lewis et al, Cell 2005,115:787-798) for potential miRNA target that 1/3rd human mRNA is arranged approximately.

More and more evidences shows, different miRNA sequences for the generation and the development of cancer play an important role (Lu et al, Nature 2005,435:834-838; Volinia et al, Proc.Natl.Acad.Sci.USA 2006,103:2257-2261; Wynter, Med.Hypotheses.2006,66:612-35; Li et al, Biochem.Biophys.Res.Commun.2006,348:229-237).The miRNA gene is usually located at the site that cancer is modified easily on the genome, fragile site (FRAs) for example, breaking point and zone; The Minimum Area of genome amplification; And the zone (Calin et al, Proc.Natl.Acad.Sci.USA.2002, the 99:15524-15529 that lose heterogeneity; Calin et al, Proc.Natl.Acad.Sci.USA.2004,101:2999-3004), more than more viewed miRNA changes of expression level are results of genomic instability in the explanation cancer.

People utilize miRNA chip express spectra (Babak et al., RNA 2004,10:1813-9; Barad et al., Genome Res.2004,14:2486-94; Calin et al., Proc Natl Acad Sci USA.2004,101:11755-11760; Liu et al., Proc Natl Acad Sci USA.2004,101:9740-4; Nelson et al., Nat Methods 2004,1:155-61; Thomson et al., Nat Methods 2004,1:47-53; Ciafre et al., Biochem.Biophys.Res.Commun.2005,334:1351-1358; Shingara et al., RNA 2005,11:1461-1470); Magnetic bead hybridization (Lu et al., Nature 2005,435:834-838) and RT-PCR (Bandres et al.; Mol Cancer 2006 5:29) studies the miRNA expression level of cancerous tissue.Found all that in many cancers the miRNA expression level changes, for example lymphocytic leukemia (Calin et al., Proc Natl Acad Sci USA.2004; 101:11755-11760), the rectum cancer (Bandres et al., Mol Cancer 2006; 5:29); Glioblastoma multiforme (Ciafre et al., Biochem.Biophys.Res.Commun.2005,334:1351-1358) and thymic carcinoma (Iorio et al.; Cancer Research 2005,65:7065-7070).At present, the cancerous tissue slicer of some preservations is like paraffin-embedded tissue (FPPE) (the Nelson et al. of Superlysoform preservation; RNA 2006,12:187-191), and stored frozen tissue (Lu et al; Nature 2005, and 435:834-838), or organizing of two kinds of each preservations of method used (He et al simultaneously; Nature 2005, and 435:828-833), or other do not offer some clarification on tissue (the Volinia et al. of preservation situation; Proc Natl Acad Sci USA.2006,103:2257-2261; Yanaihara et al., Cancer Cell 2006 9:189-198) is successfully applied to the analysis of miRNA express spectra in the cancer.

Reference is all intactly classified in all related here publications, patent, patented claim and disclosed patented claim as.

Summary of the invention

The present invention relates to state, carry out the diagnosis and the prognosis of cancer, especially for the diagnosis and the prognosis of the esophageal carcinoma according to level or the corresponding gene of miRNA.The present invention also provides diagnosis and the prognosis that application probe is used for cancer, particularly applying detection miRNA or corresponding gene state probes to be used for the diagnosis and the prognosis of the diagnosis and the prognosis, the particularly esophageal carcinoma of cancer in addition.

Corresponding therewith; The invention provides the individual cancers method of diagnosing; The method includes the steps of: a) measure at least a miRNA level in the group of individuals tissue samples; B) the miRNA level in the comparative measurement sample with reference to the miRNA level, with reference to the miRNA level obvious change is arranged relatively if the miRNA level is same in the working sample tissue, explain that individuality has canceration.

On the other hand; The invention provides diagnosing esophageal cancer method in individuality; The method includes the steps of: a) measure and suspect at least a miRNA level that the canceration part is arranged in the individual esophageal tissue sample, for example measure at least a or its corresponding homologue among the miRNA among Fig. 1, b) the miRNA level in the comparative measurement sample with reference to the miRNA level; If the miRNA level explains that with reference to the miRNA level obvious change being arranged relatively individuality has esophagus cancer in the working sample tissue.Specifically, the esophageal carcinoma that aforesaid method is measured can be esophageal squamous cell carcinoma, also can be adenocarcinoma of esophagus.

Specifically, the miRNA that in aforesaid method, measures is not miR-29b, miR-29a, miR-96, miR-182s, miR-182as, miR-183 and miR-129-1, neither miR-15 and miR-16.

Specifically, in above-mentioned esophagus cancer diagnostic method, at least one (can be 2,5 at least also for example, 10,14) be determined from miRNA level or their corresponding homologue levels of the selection that contains SEQ ID Nos.1-14; If in detection, have at least miRNA or their a corresponding homologue that contains SEQ ID Nos.1-14 that remarkable rising is arranged among the result, showing has esophagus cancer in the sample.Under the other particular case; At least one is determined from the level of the miRNA that contains SEQ ID Nos.15-38 and select or their corresponding homologues; If in detection, have at least miRNA or their a corresponding homologue that contains SEQ ID Nos.15-38 that remarkable reduction is arranged among the result, showing has esophagus cancer in the sample.Specifically; At least one miRNA that from SEQ ID Nos.1-14, selects selects from the miRNA of SEQ ID Nos.15-38 with at least one in aforesaid method; If in detection, have at least a miRNA who contains SEQ ID Nos.1-14 to have the miRNA of remarkable rising and at least one SEQ ID Nos.15-38 that remarkable reduction is arranged among the result, showing has esophagus cancer in the sample.

Specifically, the level of in aforesaid method, measuring miRNA is used the analyzing biochips method.

Specifically, the miRNA level is confirmed through the hybridization signal of miRNA on the chip in aforesaid method.Specifically in aforesaid method between the hybridization signal of miRNA level through miRNA on the chip and the reference sample hybridization signal ratio definite.Specifically the miRNA level is confirmed with the arbitrary method in Northern hybridization, in situ hybridization and the quantitative RT-polymerase chain reaction method in the aforesaid method.

Specifically, a kind of in human body the method for diagnosing esophageal cancer, this method comprises to analyze suspects the corresponding gene state of at least a miRNA, the for example corresponding gene state of the miRNA in Fig. 1 in the tissue part that canceration is arranged in esophageal tissue's sample in the human body; Compare the gene state of the miRNA in the test sample and the gene state of reference article miRNA, if the corresponding gene of the miRNA in the test sample tissue explains that with the obvious change of having compared with reference to gene among the article miRNA human body has esophagus cancer.Specifically, the gene state of measuring miRNA in the aforesaid method is confirmed through gene elmination or amplification.Specifically, the gene alteration of definite miRNA is the change according to gene copy number in aforesaid method.

Specifically, the invention provides the system of the level that is used to measure at least one miRNA as shown in Figure 1 or their corresponding analogs (perhaps corresponding miRNA gene state), for example micro-array chip; This system is made up of a plurality of probes; Each probe can detect a miRNA or the gene state of corresponding miRNA in esophageal tissue's sample; The probe of (for example comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) can detect miRNA as shown in fig. 1 or their corresponding analogs to have 15% at least, or the gene state of corresponding miRNA.Specifically, the invention provides the system that comes diagnosis of esophageal, for example a micro-array chip; This system is made up of many probes; Each probe can detect the miRNA (or gene state of corresponding miRNA) in the sample, and the probe of (for example comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) can detect miRNA as shown in fig. 1 (or gene state of corresponding miRNA) to have 15% at least.

Specifically; The invention provides system of application and come diagnosis of esophageal, this system is made up of at least one (comprising for example at least 2,5,10,15,20,25,30,35 and 40) probe, for example oligonucleotide; Each probe can detect miRNA level as shown in fig. 1 or the gene state of corresponding miRNA; If miRNA level as shown in fig. 1 or their respective analogs, or in the gene state of corresponding miRNA at least one have obvious change, and the esophageal carcinoma has been described.Specifically; The invention provides application system and come diagnosis of esophageal; Micro-array chip for example; This system is made up of many probes, and each probe can detect the level of the gene state of miRNA in the sample or corresponding miRNA, and the probe of (for example comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) can detect miRNA as shown in fig. 1 or their corresponding analogs wherein to have 15% at least; Or the level of the gene state of corresponding miRNA, if the level of the gene state of miRNA as shown in fig. 1 or their corresponding analogs or corresponding miRNA has considerable change to show the esophageal carcinoma is arranged.

The present invention also provides the probe that detects miRNA to be used to prepare said system.Specifically; The invention provides with one or more probes (for example oligonucleotide) and make the system that is used to diagnose the individual esophageal carcinoma; Each probe can detect miRNA as shown in fig. 1 or their corresponding analogs, or the gene status level of corresponding miRNA.Specifically; The present invention makes the system's (for example micro-array chip) in order to diagnosis of esophageal with probe (for example oligonucleotide); Each probe can detect the level of a miRNA in the sample or the gene state of corresponding miRNA; The probe of (for example comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) can detect miRNA as shown in fig. 1 or their corresponding analogs to have 15% at least, or the gene status level of corresponding miRNA.

On the other hand, the present invention also provides the method for patient with esophageal carcinoma somatotype, for example confirms level of differentiation, neoplasm staging and the histological type of esophagus cancer.Specifically; The invention provides the method for dividing esophageal carcinoma patient; Comprise and for example judge level of differentiation, neoplasm staging and histological type that this method comprises at least one miRNA in the individual human esophageal carcinoma of detection, miRNA that example is as shown in table 2 or their corresponding analogs; Or the gene state of corresponding miRNA, and the gene state of the level of miRNA or corresponding miRNA is as the basis of dividing esophageal carcinoma patient.

Specifically; The invention provides the method for the individual esophageal carcinoma level of differentiation of decision; Comprise the miRNA shown in Fig. 2 a or the level of their corresponding analogs of detecting in the individual human esophageal carcinoma; Or the gene state of corresponding miRNA, and the gene state of the level of miRNA or corresponding miRNA is the basis as the individual esophageal carcinoma level of differentiation of decision.Specifically, the miRNA of detection at least one be hsa-miR-335 or its corresponding analogs.In some cases, at least one miRNA is hsa-miR-25 or its corresponding analogs.Specifically, the miRNA of detection at least one be hsa-miR-130b or its corresponding analogs.Specifically, the miRNA of detection at least one be hsa-miR-130a or its corresponding analogs.Specifically, the miRNA of detection at least one be hsa-miR-181d or its corresponding analogs.

Specifically; The invention provides a system and detect at least one miRNA as shown in table 2 or their corresponding analogs in the sample; The level of perhaps corresponding miRNA gene state; Microarray system for example; This system includes a plurality of probes, and each probe can detect a miRNA or the gene state of corresponding miRNA in the sample, and the probe of (for example comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) can detect miRNA as shown in table 2 or their corresponding analogs (or gene state of corresponding miRNA) to have 15% at least.Specifically, the invention provides through a system and divide patient with esophageal carcinoma, for example microarray; This system is made up of a plurality of probes; Each probe can detect a miRNA or the gene status level of corresponding miRNA in the sample, and have 15% at least the probe of (for example comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) can detect the level of miRNA as shown in table 2 or their corresponding analogs (or gene state of corresponding miRNA).

Specifically, the invention provides system of application and divide patient with esophageal carcinoma, this system is made up of one or more probes, and each probe can detect the level of miRNA as shown in table 2 or their corresponding analogs, or the gene state of corresponding miRNA.Specifically; The invention provides system of application and divide patient with esophageal carcinoma; System is made up of one or more probes, and each probe can detect a miRNA in the sample, and at least 50% probe can detect the level of miRNA as shown in table 2 or their corresponding analogs.

Specifically, the invention provides the one or more probes of application and make in order to divide the system of patient with esophageal carcinoma, each probe can detect the level of miRNA as shown in table 2 or their corresponding analogs, or the gene state of corresponding miRNA.Specifically, the invention provides application probe makes in order to divide the system of patient with esophageal carcinoma, for example microarray; Each probe can detect the level of a miRNA in the sample or the gene status level of corresponding miRNA, and have 15% at least the probe of (for example comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) can detect the level of miRNA as shown in table 2 or their corresponding analogs (or gene state of corresponding miRNA).

Specifically, native system is applied to confirm the level of differentiation of individual patient with esophageal carcinoma esophagus cancer.Specifically, native system is applied to divide squamous cell carcinoma of esophagus from pathology.Specifically, native system can be applied to confirm the neoplasm staging of the individual esophageal carcinoma.

The present invention provides about judging the method for patient with esophageal carcinoma prognosis in addition on the one hand, comprises the existence prognosis of judging patient with esophageal carcinoma.Specifically; The existence method of prognosis of judging patient with esophageal carcinoma is provided; The method includes the steps of: a) detect the level of at least one miRNA in the individual human esophageal carcinoma sample, b) level and the threshold value of this miRNA are compared, this ratio becomes positive correlation or negative correlation with individual survival rate.Specifically; The invention provides the method for the existence prognosis of judging patient with esophageal carcinoma; Comprise at least one miRNA gene state of analysis; For example one gene state among hsa-miR-103, hsa-miR-107 and the hsa-miR-23b is compared changing of gene state patient's high survival rate or low survival rate is described with check sample.

Specifically, at least one is one or their corresponding analogs among hsa-miR-103, hsa-miR-107 or the hsa-miR-23b among the above-mentioned miRNA.Specifically, at least one miRNA is hsa-miR-107.Specifically, at least one miRNA is hsa-miR-23b.Specifically, survival rate is total survival rate.Specifically, survival rate is the DFS rate.Specifically, to be in the esophageal carcinoma early stage for individuality.Specifically, this method also comprises the suitable course of treatment that decision is individual.

The present invention also provides the probe of using the gene state that can detect miRNA level or corresponding miRNA, or use the system that includes one or more probes be used for judging the existence prognosis.Specifically; The present invention provides one or more probes or includes the existence prognosis that patient with esophageal carcinoma is judged by the system that is made up of one or more probes; Each probe can detect a miRNA in the sample; Level and the threshold value of this miRNA are compared, and this ratio becomes positive correlation or negative correlation with this individual survival rate.Specifically; The invention provides and use the existence prognosis that one or more probes are judged patient with esophageal carcinoma; Level and the threshold value of miRNA are compared; This ratio and this individual survival rate negative correlation, these miRNA have at least one to be hsa-miR-103, hsa-miR-107 or hsa-miR-23b or their corresponding analogs.

Specifically; The invention provides the one or more probes of application and make reagent (or system) in order to prediction patient with esophageal carcinoma existence prognosis; Each probe can detect the miRNA in the sample; Level and the threshold value of this miRNA are compared, and this ratio becomes positive correlation or negative correlation with this individual survival rate.Specifically; Provide with one or more probes and made reagent (or system) in order to prediction patient with esophageal carcinoma existence prognosis; MiRNA level and critical ratio and this individual survival rate negative correlation, such miRNA has at least one to be hsa-miR-103, hsa-miR-107 or hsa-miR-23b or their corresponding analogs.Specifically, having a miRNA at least is hsa-miR-103, hsa-miR-107 or hsa-miR-23b.

The present invention provides the method that improves the patient with esophageal carcinoma survival rate simultaneously.Specifically, the invention provides the method that improves the patient with esophageal carcinoma survival rate, this method comprises reagent, the ratio of this miRNA level and threshold value and this individual survival rate negative correlation that the significant quantity that reduces certain miRNA level is provided to the patient.Specifically, the invention provides the application of reagent in the preparation medicine that reduces certain miRNA level, this medicine is used for improving the patient with esophageal carcinoma survival rate, the ratio of this miRNA level and threshold value and this individual survival rate negative correlation.

Specifically; The invention provides the method that improves the patient with esophageal carcinoma survival rate; This method comprises that miRNA comprises hsa-miR-103, hsa-miR-107, hsa-miR-23b or their corresponding analogs to reducing the reagent of certain miRNA level with patient's effective dose.Specifically, the present invention also provides and has used the reagent that reduces certain miRNA level, in the medicine of preparation raising patient with esophageal carcinoma survival rate, uses, and this type of miRNA comprises hsa-miR-103, hsa-miR-107, hsa-miR-23b or their corresponding analogs.Specifically, these reagent reduce by two kinds among hsa-miR-103, hsa-miR-107 or the hsa-miR-23b at least simultaneously.Specifically, these reagent can reduce hsa-miR-103, hsa-miR-107 and hsa-miR-23b simultaneously.

Specifically; The invention provides and contain the reagent that can reduce a kind of miRNA level at least and the medicinal composition of the applicable carrier of a kind of medicine, the miRNA that this medicinal composition can reduce comprises at least a in hsa-miR-103, hsa-miR-107 or hsa-miR-23b or their corresponding analogs.Under some situation, at least one miRNA is hsa-miR-103.Specifically, at least one miRNA is hsa-miR-107.Specifically, at least one miRNA is hsa-miR-23b.Specifically, this kind medicine is a for example ribozyme of double-stranded RNA (for example short or little RNA interfering, or siRNA), GEM 132 or the RNA molecule with enzymic activity.

The present invention also comprises the test kit that above-mentioned the whole bag of tricks is provided.

Description of drawings

Fig. 1 miRNA that level changes in patient with esophageal carcinoma.

Fig. 2 a miRNA that level changes in the patient with esophageal carcinoma of different differential periods.

Fig. 2 b is at the miRNA that level changes in cap type and the medullary squamous cell carcinoma of esophagus

Fig. 2 c miRNA that level changes in the patient with esophageal carcinoma of different tumor stages (N0/N1).

The miRNA of Fig. 3 expression level and esophageal carcinoma survival rate negative correlation.

The cluster analysis of the miRNA that Fig. 4 picks out with microarray significance analysis (SAMs) method.40 miRNA differential expression in freezing human esophageal carcinoma and cancer beside organism is arranged, the express spectra of these 40 miRNA in 31 pairs of sample groups is carried out cluster analysis.Sample is arranged by row, and miRNA by rows.The sample that indicates # is represented " mis-classification ".

Fig. 5 has compared the miRNA express spectra of flesh tissue and frozen tissue.The miRNA express spectra is through 5 pairs of flesh tissue checkings.These 10 samples are used the SVM method validation, confirm all correct classification of 10 samples.Indicate the learning sample prompting " mis-classification " of #.

Fig. 6 is the Kaplan-Meier survival curve of patient with esophageal carcinoma.The low expression (n=15) of Fig. 6 A prompting hsa-miR-103 and total survival rate positive correlation of patient with esophageal carcinoma, total survival rate negative correlation of the high expression level of hsa-miR-103 (n=16) and patient with esophageal carcinoma.The low expression (n=14) of Fig. 6 B prompting hsa-miR-107 and total survival rate positive correlation of patient with esophageal carcinoma, total survival rate negative correlation of the high expression level of hsa-miR-107 (n=17) and patient with esophageal carcinoma.The low expression (n=17) of figure C prompting hsa-miR-23b and total survival rate positive correlation of patient with esophageal carcinoma, total survival rate negative correlation of the high expression level of hsa-miR-23b (n=14) and patient with esophageal carcinoma.The low expression (n=15) of figure D prompting hsa-miR-103 and the DFS rate positive correlation of patient with esophageal carcinoma, the DFS rate negative correlation of the high expression level of hsa-miR-103 (n=16) and patient with esophageal carcinoma.The low expression (n=14) of figure E prompting hsa-miR-107 and the DFS rate positive correlation of patient with esophageal carcinoma, the DFS rate negative correlation of the high expression level of hsa-miR-107 (n=17) and patient with esophageal carcinoma.

Embodiment

The present invention is mainly based on the miRNA express spectra research of the genomic level that 31 pairs of normal esophageal tissues and human esophageal carcinoma are carried out.Specifically, promptly be to compare the miRNA differential expression in cancerous tissue and the corresponding adjacent tissues with micro-array chip.We find 40 high expression level or the low miRNA that express in cancerous tissue.We have also found and have expressed discrepant miRNA under the various disease state.In addition, we find that also the level of three miRNA is relevant with the total survival rate and the DFS rate of patient with esophageal carcinoma.

The present invention is based on the expression level of some miRNA or the state of corresponding gene, for the diagnosis and the prognosis of cancer (the for example esophageal carcinoma) provides method and medicine.

From an aspect, we provide the relevant cancer (the for example esophageal carcinoma) based on some miRNA expression level method of diagnosing and medicine.

From another aspect, we also provide based on some miRNA expression level, the method for dividing cancer patients, especially patient with esophageal carcinoma according to histological type, level of differentiation and neoplasm staging.

From another aspect, we also provide based on some miRNA expression level, judge the method and the medicine of the existence prognosis of cancer patients, especially patient with esophageal carcinoma.

From an aspect, we provide the method that detects the miRNA expression level with RT-PCR, in order to diagnose the illness.

The present invention provides the system and the test kit of above-mentioned all methods.

" individuality " expression vertebrates, especially a Mammals, particularly people.Mammals not only refers to domestic animal, wild poultry, pet, primates, mouse and rat.In some cases, individuality is meant the people.In some cases, individuality is meant the model animal in order to the research esophageal carcinoma.That is to say that if individual that refer to is not the people, miRNA just is meant the corresponding analogs or the homologue of corresponding human miRNAs so.

In some cases, individuality is meant the male sex.In some cases, individuality is meant the women.In some cases, the individual histological type of not representing the esophageal carcinoma.In some cases, individuality has been meant esophageal carcinoma family history.

Here said one " esophageal tissue's sample " is meant the tissue samples of oesophagus.In some cases, tissue samples is a flesh tissue.In some cases, tissue samples is a frozen tissue.In some cases, tissue samples is fixed.In some cases, tissue samples is fixed by formaldehyde.In some cases, tissue samples is by paraffin embedding.Be described below,, use complete tissue, perhaps use the tissue that is divided into fritter or cell mass or individual cells according to concrete method.

The esophageal carcinoma includes, but are not limited to squamous cell carcinoma of esophagus and adenocarcinoma of esophagus.

In order to diagnosing cancer, the particularly method of the esophageal carcinoma and medicine

From an aspect, the present invention is that the method that individual disease (for example cancer) diagnosis provides comprises: a) detect the level from least one miRNA of certain individual specimen; B) level and the contrast of this miRNA are compared, the typical change of miRNA level can be used as the prompting of disease (for example cancer).

The disease that can diagnose with the present invention comprises: cancer, for example lung cancer, mammary cancer, the esophageal carcinoma, cancer of the stomach, liver cancer, large bowel cancer, carcinoma of the pancreas, white blood disease, lymphoma, kidney, bladder cancer, cervical cancer, carcinoma of endometrium, ovarian cancer, carcinoma of testis; Cardiovascular disorder, for example coronary heart disease, hypertension, arteriosclerosis; Age related disease, for example parkinson's disease, sick, the mellitus of A Erci Mohs.

From an aspect, the present invention is that the method that esophagus cancer diagnosis provides comprises: a) detection is from the level of at least one miRNA (at least one miRNA that for example shows among Fig. 1 or their corresponding homologues) of esophageal tissue's sample of certain esophageal carcinoma suspected case; B) with the level of this miRNA with reference to comparing, the characteristic change of miRNA level can be used as the prompting of the esophageal carcinoma.

In some cases, the present invention is that the method that esophagus cancer diagnosis provides comprises: a) will be from miRNA (at least one miRNA that for example shows among Fig. 1 or their the corresponding homologues) level of esophageal tissue's sample of certain esophageal carcinoma suspected case with reference to comparing; B) judge according to the characteristic change of at least one miRNA level whether individuality suffers from the esophageal carcinoma.In some cases, method also comprises the step that obtains esophageal tissue's sample from individuality.In some cases, method also comprises the step of extracting miRNA from tissue samples.

In some cases, the present invention is that the method that esophagus cancer diagnosis provides comprises: a) detection is from the level of at least one miRNA (at least one miRNA that for example shows among Fig. 1 or their corresponding homologues) of esophageal tissue's sample of certain esophageal carcinoma suspected case; B) level according to this miRNA is that esophagus cancer diagnosis provides information, and the level of this miRNA can be used as diagnostic base, and the characteristic change of this miRNA level can be used as the prompting of the esophageal carcinoma.

In some cases, miRNA is not miR-29b, miR-29a, and miR-96, miR-182*, miR-182a*, miR-183, and miR-129-1, in some cases, miRNA is not miR-15 and miR-16.

In some case, detect the level of at least one miRNA (for example being in 2,5,10 at least) in SEQ ID numbering 1-14 or the corresponding analogs, wherein the remarkable increase of at least one miRNA level can be pointed out the esophageal carcinoma.In some case, detect the level of at least one miRNA (for example being in 2,5,10,15,20,24 at least) in SEQ ID numbering 15-38 or the corresponding analogs, wherein the remarkable increase of at least one miRNA level can be pointed out the esophageal carcinoma.In some case; At least one miRNA (for example being in 2,5,10,14 at least) at least one miRNA (for example being in 2,5,10,14 at least) and SEQ ID numbering 15-38 or the corresponding analogs in detection SEQ ID numbering 1-14 or the corresponding analogs; Wherein have at least a miRNA level significantly to increase in miRNA and their corresponding analogs of numbering 1-14 among Fig. 1, number simultaneously in miRNA and their corresponding analogs of 15-38 and have at least the remarkable decline of miRNA level can point out the esophageal carcinoma.

In some case; We detect the level of all miRNA shown in Figure 1; The level of at least one miRNA (for example being in 2,5,10 at least) raises among the SEQ ID numbering 1-14, and the level decline of at least one miRNA (for example being in 2,5,10,14 at least) can be pointed out the esophageal carcinoma among the SEQ ID numbering 15-38 simultaneously.In some cases, the level of at least two miRNA raises among the SEQ ID numbering 1-14, and the level decline of at least two miRNA can be pointed out the esophageal carcinoma among the SEQ ID numbering 15-38 simultaneously.In some cases, the level of the miRNA among the SEQ ID numbering 1-14 raises, and the level decline of the miRNA among the SEQ ID numbering 15-38 simultaneously can be pointed out the esophageal carcinoma.

The level of miRNA can reflect the variation of miRNA gene state in the tissue samples.The change of gene state can be reflected or changed by the copy number of miRNA gene and reflect by miRNA genetically deficient or amplification.

Therefore in some cases; The method that we provide for esophagus cancer diagnosis comprises at least one miRNA (at least one miRNA of for example in Fig. 1 showing or their corresponding homologue) the gene state of analysis from the tissue samples of esophageal carcinoma suspected case, if can point out the esophageal carcinoma than the change of check sample producer state.In some cases, the change of gene state is decided by miRNA genetically deficient or amplification, and in some cases, the change of gene state is decided by the copy number variation of miRNA gene.

In some cases; The method that we provide for esophagus cancer diagnosis comprises that analysis is from the miRNA shown at least one Fig. 1 of the tissue samples of esophageal carcinoma suspected case; See whether this miRNA gene has disappearance or amplification, if can point out the esophageal carcinoma than check sample producer disappearance or amplification.For example, in some cases, whether the miRNA gene that we analyze among the SEQ ID numbering 1-14 increases, and the amplification of at least one miRNA gene can be pointed out the esophageal carcinoma.In some cases, whether the miRNA gene that we analyze among the SEQ ID numbering 15-38 lacks, and the disappearance of at least one miRNA gene can be pointed out the esophageal carcinoma.In some cases, method also comprises the step that obtains esophageal tissue's sample from suspected case.In some cases, method also comprises the step of extracting DNA from tissue samples.

In some cases, the method that we provide for esophagus cancer diagnosis comprises in the tissue samples of analysis from esophageal carcinoma suspected case whether the variation of the miRNA gene copy number shown in Fig. 1 is arranged.If have on women's euchromosome or the sex chromosome, can point out the esophageal carcinoma if having on the male sex chromosome more than a miRNA gene more than two miRNA genes.For example; In some cases; We analyze the copy number of the miRNA gene among the SEQ ID numbering 1-14, if at least one miRNA gene has on women's euchromosome or sex chromosome more than two copy numbers, have on the male sex chromosome more than a copy number and can point out the esophageal carcinoma.In some cases; We analyze the copy number of the miRNA gene among the SEQ ID numbering 15-38; If at least one miRNA gene is less than two copy numbers on women's euchromosome or sex chromosome, is less than a copy number on the male sex chromosome and can points out the esophageal carcinoma.In some cases, method also comprises the step that obtains esophageal tissue's sample from suspected case.In some cases, method also comprises the step of extracting DNA from tissue samples.

Here described miRNA has following effect: level and an above miRNA gene state according to an above miRNA in the human esophageal carcinoma sample are divided the progress of cancer of esophagi people type, the prediction esophageal carcinoma, monitoring esophageal carcinoma patient's the course of disease and monitoring esophageal carcinoma patient's treatment.

In some situation, we provide the system's (for example microarray) that is used for detecting miRNA as shown in Figure 1 or their corresponding analogs level, or system's (for example microarray) of the gene state that is used for detecting miRNA as shown in Figure 1 or their corresponding analogs is provided.These systems are very useful for judging miRNA as shown in Figure 1 or their corresponding analogs level and diagnosis of esophageal.Tell about system below emphatically, but, also can on top of detect the system of miRNA gene state for the personnel that are familiar with this generic operation in order to detection miRNA level.

In some situation; We judge the level of at least one miRNA as shown in Figure 1 or their corresponding analogs (perhaps corresponding miRNA gene state) through a system (for example microarray); System is made up of many probes; Each probe can detect the miRNA (or gene state of corresponding miRNA) in esophageal tissue's sample, and the probe of (for example comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) can detect miRNA as shown in fig. 1 or their corresponding analogs (or gene state of corresponding miRNA) to have 15% at least.In some situation; We come diagnosis of esophageal through a system (for example microarray); System is made up of many probes; Each probe can detect the miRNA (or gene state of corresponding miRNA) in the sample, and the probe of (for example comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) can detect miRNA as shown in fig. 1 (or gene state of corresponding miRNA) to have 15% at least.In some situation; We come diagnosis of esophageal through a system (for example microarray); System is made up of at least one (comprising for example at least 2,5,10,15,20,25,30,35 and 40) probe (for example oligonucleotide), and each probe can detect miRNA level as shown in fig. 1 (or gene state of corresponding miRNA).To describe these systems (for example microarray) below in detail.

Can there be multiple effect in the system of these diagnosis of esophageal.In some situation; We come diagnosis of esophageal through a system; System is made up of at least one (comprising for example at least 2,5,10,15,20,25,30,35 and 40) probe (for example oligonucleotide); Each probe can detect miRNA level as shown in fig. 1 (or gene state of corresponding miRNA), thereby the horizontal properties property variation of at least one can be pointed out the esophageal carcinoma in miRNA as shown in fig. 1 or their respective analogs (or gene state of corresponding miRNA).In some situation; We come diagnosis of esophageal through a system (for example microarray); System is made up of many probes; Each probe can detect the level of a miRNA (or gene state of corresponding miRNA) in the sample; Thereby have 15% at least the probe of (for example comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) can detect the level of miRNA as shown in fig. 1 or their corresponding analogs (or gene state of corresponding miRNA), and the horizontal properties property variation of miRNA as shown in fig. 1 or their corresponding analogs (or gene state of corresponding miRNA) can be pointed out the esophageal carcinoma.

It below is description about the probe of the used detection miRNA of manufacturing system.In some situation, we make in order to diagnose the system of the individual esophageal carcinoma with one or more probes (for example oligonucleotide), and each probe can detect the level of miRNA as shown in fig. 1 or their corresponding analogs (or gene state of corresponding miRNA).In some situation; We make the system's (for example microarray) in order to diagnosis of esophageal with probe (for example oligonucleotide); Each probe can detect the level (or gene state of corresponding miRNA) of a miRNA in the sample, and have 15% at least the probe of (for example comprise at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) can detect the level of miRNA as shown in fig. 1 or their corresponding analogs (or gene state of corresponding miRNA).

The miRNA that diagnosis of esophageal is related

40 miRNA that level is relevant with the esophageal carcinoma have been confirmed in existing invention.Like table 2 and shown in Figure 1.Fig. 1 provides title, sequence and the chromosomal localization of these miRNA.Can pass through the information that http://miRNA.sanger.ac.uk/. (referring to Griffths-Jones et al., Nucleic Acids Research, 2006, Vol.34, Database issue) obtains these miRNA.The method of diagnosis of esophageal is based on miRNA level shown in Figure 1 and gene state.Here described system is used for monitoring miRNA level shown in Figure 1, and then diagnosis of esophageal.

Through practice, can confirm accepted sensitivity and the specificity of these methods, yet the validity (for example sensitivity and specificity) of method will improve greatly when detecting two above miRNA in order to detect single miRNA.For example, in some cases, to detect 3,4 among Fig. 1 at least, 5,6,7,8,9,10,15,20,25,30,35 or No. 38 miRNA.

In some cases, we detect two miRNA (for example being in 3,5,10 at least) among the SEQ ID numbering 1-14 at least.In some cases, we detect two miRNA (for example being in 3,5,10,15,20 at least) among the SEQ ID numbering 15-38 at least.In some cases, we detect the gene state that miRNA and SEQ ID among the SEQ ID numbering 1-14 numbers a miRNA among the 15-38 at least.In some cases, we detect the gene state that two miRNA (for example being in 3,5,10 at least) and SEQ ID among the SEQ ID numbering 1-14 numbers two miRNA (for example being in 3,5,10,15,20 at least) among the 15-38 at least.In some cases, we detect the gene state of all miRNA among Fig. 1.

In some cases, the level that needs the corresponding analogs of the above-mentioned miRNA of judgement." corresponding analogs " of above-mentioned miRNA refers to the miRNA (also comprising at least 60%, 70%, 80%, 90%, 95%, 98% and 99% sequence similarity) that has at least 50% sequence similarity with miRNA.For example, the corresponding analogs of SEQ ID NO:1 miRNA has the sequence similarity (also comprising at least 60%, 70%, 80%, 90%, 95%, 98% and 99% sequence similarity) with SEQ ID NO:1 miRNA at least 50%.

The miRNA that has 95% above similarity with reference sequences (SEQ ID NO:1 miRNA) can think identical with reference sequences, only if per 100 Nucleotide have 5 point mutation.These 5 point mutation can be disappearance, replacement, insert, and can be dispersed in and perhaps concentrate on one or more bunches of reference sequences in the reference sequences in the generation Anywhere of sequence.

Esophagus cancer diagnosis method based on the miRNA level

In some cases, the esophagus cancer diagnosis method is based on the miRNA level.

Here said " level " is meant amount or the speed that miRNA or its precursor are piled up.This speech can refer to the absolute magnitude (for example representing with hybridization signal intensity) of miRNA in certain sample, also can refer to miRNA amount and the ratio that contrasts (for example comparing the hybridization signal ratio of photograph with sample) in the sample.Contrast miRNA can be the different miRNA of the relative constant of level in the same sample, also can be the same miRNA (for example from the non-cancer tissue sample of same individuality, perhaps the individual tissue samples of another non-esophageal carcinoma) in the different samples.

The precursor of miRNA molecule or " miRNA precursor " refer to miRNA genetic transcription of not shearing, and typically comprise rna transcription of about 70 length of nucleotides.The miRNA precursor is become active miRNA molecule by RNA enzyme (for example Dicer, Argonaut or RNA enzyme III) digestion, is typically 19-25 length of nucleotides.

" the miRNA level of esophageal tissue's sample " is meant the miRNA level of tissue samples.In most of the cases; The miRNA level of esophageal tissue's sample is to obtain through the direct miRNA level of measuring esophageal tissue's sample, yet the miRNA level of esophageal tissue's sample also can reflect through lymphoglandula (for example contiguous lymphoglandula or lymph liquid) sample, blood plasma, whole blood or contiguous body fluid such as sputum.In some cases, the miRNA level is that miRNA level through lymphoglandula sample (for example lymphoglandula work is cut or pin inhale) decides.In some cases, the miRNA level is to decide through the miRNA level in whole blood or the blood plasma.In some cases, the miRNA level is that the miRNA level that esophageal tissue draws in the net decides.In some cases, the miRNA level is to decide through the miRNA level (for example RT-PCR analyzes) in the endoscope guiding sampling gained sample.The pin of endoscope guiding is inhaled (FNA) sampling and is the minimum technology of invasive, and is especially suitable for the non-operation property sampling of mediastinal lymph nodes, and can carry out more detailed molecular marker analysis.MiRNA horizontal analysis to non-human esophageal carcinoma can be united use separately and with other method.For example, can at first confirm the miRNA level in the blood plasma, the miRNA level of analyzed area lymphoglandula then, the rapid analysis of multistep can provide abundanter information and increase the confidence level of diagnosis like this.

Can detect the miRNA level in different periods.For example, can be before art, in the art, postoperative, and before the oncotherapy, in the treatment, the treatment back detects the miRNA level.In some cases, the miRNA level is that miRNA level in the sample (particularly esophageal tissue) that is drawn in the net to be obtained by esophageal tissue decides.

The method of measuring the miRNA level has report in document.For example, Northern dot hybridization, in situ hybridization, RT-PCR and microarray.These methods have report in document, Einat for example, Methods Mol.Biol.2006,342:139-157; Thompson et al., Genes Dev.2006,20:2202-2207.

According to classical way, total RNA of cell can be through kytoplasm homogenization in the environment of nucleic acid extraction damping fluid and then centrifugal obtaining.Nucleic acid is precipitated, and DNA removes through the centrifugal again back of DNA enzyme effect.RNA separates with gel electrophoresis through standardisation technique, transfers to nitrocellulose filter again, carries out Northern dot hybridization etc.RNA is fixed on the film through heating.Through identifying with quantitative with fluorescently-labeled DNA of target RNA complementary or rna probe.On one-tenth phase film, also can identify miRNA through the radioautograph method.Be scanned into the phase film, can be to the horizontal accurate quantification of rna transcription.Also can carry out the rna level accurate quantification to hybridization spot with light tomography computer method of calculation.

Except Northern and other dot hybridization technology, the sub-level of rna transcription can also be measured through in situ hybridization.This technology is that whole cell or tissue is fixed on the cover glass, utilizes the solution of the probe contain the radioactive rays mark or other mode label probes (for example cRNA probe) to measure the nucleic acid of cell or tissue.

The miRNA level also can be carried out polymerase chain reaction,PCR (RT-PCR) and detect through to miRNA transcripton rt then.The level of miRNA can be through comparing quantitatively with confidential reference items, and confidential reference items can be the mRNA of house-keeping gene in the same sample etc.Suitable house-keeping gene as confidential reference items comprises myosin, 3` phosphoglyceraldehy-de dehydrogenase or people U6 gene.Quantitative RT-PCR and other description of related art are arranged in document.Table 1 has been listed the primer commonly used of RT-PCR.In some cases, real-time RT-PCR (qRT-PCR) detection miRNA cuts than classical tissue work or is sensitive more in the early stage miRNA dyeing of cancer detection method.QRT-PCR detection miRNA level is sensitiveer and special method for diagnosis, classification and the prognosis evaluation of the esophageal carcinoma.Existing invention provides the method that detects individuality (suffering from disease, for example the individuality of cancer) miRNA level with RT-PCR.In some cases, the miRNA level detects with the qRT-PCR method.

In some cases, the miRNA level detects with the microarray of this literary composition description.

Nucleic probe described in the aforesaid method can obtain with the method for vitro recombination or chemosynthesis, and this is existing report in document.In addition, hybridization probe can come mark with the different markers method, for example ri, fluorescent substance, reporting system enzyme, vitamin H and other parts.The all right coupling optical detection material of these detectable affinity tags, and then can detect with photochemical method.The probe of mark and detection also has report in document.

Being used for detecting the nucleic probe of miRNA can be under stringent condition and the miRNA hybridization of sample.According to diverse ways, the mature technology in the document can change condition, thereby optimizes the detection to specific miRNA in the sample-specific.

Generally speaking, the stability of hybrid depends on ionic concn and temperature.In general, hybridization carries out under the condition of low preciseness, under the condition of high preciseness, washs then.Appropriateness preciseness hybridization is meant for example probe and the complementary nucleic acid molecule condition of hybridizing of nucleic acid molecule that allows.The nucleic acid molecule of hybridization has 60% similarity at least, also comprises 70%, 75%, 80%, 85%, 90%, or 95% similarity.Appropriateness preciseness hybridization conditions is equivalent to 42 ℃ hybridizes under 50% methane amide, 5xDenhart`s solution, 5xSSPE, 0.2%SDS condition, and 42 ℃ with 0.2xSSPE, 0.2%SDS washing then.High preciseness hybridization conditions can be 42 ℃ hybridizes under 50% methane amide, 5xDenhart`s solution, 5xSSPE, 0.2%SDS condition, and 65 ℃ with 0.1xSSPE, 0.1%SDS washing then.

Low preciseness hybridization is meant under the hybridization conditions suitable with following condition hybridizes: 10% methane amide, 5 * Denhart ' s solution, 6 * SSPE, 0.2%SDS, 22 ℃ of hybridization are cleaned with 1 * SSPE, 0.2%SDS at 37 ℃ then.Denhart ' s solution contains 1%Ficoll, 1% Vilaterm pyrogallol and 1% Ox blood serum.20 * SSPE (sodium-chlor, sodium phosphate, EDTA) contains 3M sodium-chlor, 0.2M sodium phosphate and 0.025M EDTA.Moderate preciseness that other are suitable and high preciseness hybridization solution and condition all for this reason the people in the field know; They also reported, for example: Sambrook et al, Molecular Cloning:A Laboratory Manual; 2nd ed; Cold Sping Harbor Press, Plainview, N.Y. (1989); And Ausubel et al., supra, 1999).

Specifically, the level of miRNA is obtaining from a patient more than a time point.The method of this " successive " sampling is well suited for keeps watch on the cancer development of patient with esophageal carcinoma.Serial sampling can be carried out at the different time point as required, as every half a year, every year, every two years or the longer time sampling once.Level that measures and the comparison between the reference level are carried out after can measuring a fresh sample at every turn, also can obtain to carry out after certain take off data again.

Method described here, reference level refer to the level that certain miRNA is considered to " normally " usually.Sometimes, reference level are meant the miRNA level in same individual's the non-human esophageal carcinoma.Sometimes, be meant the people's who does not have the esophageal carcinoma level.Be meant that sometimes a group does not have the people's of the esophageal carcinoma the M.L. of level.Sometimes, reference level come from sample library, comprise sample itself.Reference level can determine also can in measure sample, determine in advance.

Value in " reference " of this use can be an absolute value, and relative value has the value of the upper limit or lower limit, a sequential value, and MV, intermediate value, intermediate value is perhaps with specific contrast or benchmark value value relatively.

With the comparison of reference value can be among Fig. 11,2,3,4,5,6,7,8,9,10,15,20,25,30,35, or 38 any miRNA or their homologue.Comparing the process of a miRNA level and reference level can carry out according to the type method easy to use that detects miRNA value in the tissue.For example, when the hybridization signal through miRNA detected the miRNA level, the comparison of level possibly compared the power of hybridization signal qualitatively through naked eyes.For detection by quantitative, relatively possibly pass through observed data, the connotation of seeing clearly the data representative carry out (as, observed data is used diagrammatic representation, such as histogram, linear graph).Process relatively can be that manual (method as through specialty is carried out visual observation) also can be automatic.

Sometimes, contrast is (for example, relatively the difference of " ratio " or per-cent) that the order of magnitude through level that compare and measure and reference carries out.At this, it is different with the order of magnitude between the reference value that " ratio " refers to the miRNA observed value of numeral description.

In the miRNA level one " distinctive change " possibly to be that the miRNA level is significant among the patient the relative reference level reduce or increase." significant increase " referred to herein as the miRNA level increases by 5% at least, for example comprises at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% or more.Similarly, " significant reduce " refers to the miRNA level and reduces 5% at least, for example comprises at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% or more.

What Fig. 1 showed is the summary of the miRNA change of expression.The change of miRNA horizontal properties property is to be used for the basis of diagnosis of esophageal.For example, sometimes, the level of at least one miRNA has been confirmed in SEQ ID Nos.1-14, and at least one significant increasing of the miRNA level of measurement just hints that the possibility of suffering from the esophageal carcinoma is arranged.Sometimes, the level of at least one miRNA has been confirmed in SEQ ID Nos.15-38, and at least one significant minimizing of the miRNA level of measurement just hints that the possibility of suffering from the esophageal carcinoma is arranged.Sometimes; When the level of at least one miRNA among the SEQ ID Nos.1-14 confirmed with SEQ ID Nos.15-38 in the level of at least one miRNA confirmed, have at least among the SEQ ID Nos.1-14 one significant increase with SEQ ID Nos.15-38 in have at least one to reduce the possibility that hint just has the trouble esophageal carcinoma significantly.

Sometimes all miRNA levels have all determined among Fig. 1, have at least among the SEQ ID Nos.1-14 and have a significant possibility that the trouble esophageal carcinoma is arranged with regard to hint that reduces among a significant increase and the SEQ ID Nos.15-38 at least.Sometimes, have at least among the SEQ ID Nos.1-14 two significant increase with SEQ ID Nos.15-38 in have at least two to reduce the possibilities that hint just has the trouble esophageal carcinoma significantly.

Under those situation, when the level of using these miRNA more than a miRNA and inconsistent hint suffered from the esophageal carcinoma, the indication of " great majority " can be considered to detected result.For example, when using 5 miRNA, wherein 3 hints have the esophageal carcinoma, and the result can think that the hint this person is diagnosed as the esophageal carcinoma.Yet in some cases, the diagnosis of the esophageal carcinoma needs at least one or the more how special distinctive variation of miRNA.For example, suppose that a miRNA is has-miR-16, the significant increase of has-miR-16 level possibly be the prerequisite of esophagus cancer diagnosis.

Diagnostic method based on the miRNA gene level

In some cases, the level of gene in patient's sample based at least a miRNA among Fig. 1 or their homologue can provide multiple esophagus cancer diagnosis method.

Sometimes, the assessment of gene level is to hint then that through analyzing the deletion or the amplification of at least one miRNA gene in sample, in the miRNA gene, detecting deletion or increase with respect to control sample this patient has the possibility of suffering from the esophageal carcinoma.

The deletion of miRNA gene or amplification can be through detecting the structure or the sequence of gene in the human esophageal carcinoma cell of suffering from carcinoma of esophagus patients under a cloud, then with the gene structure or the sequence contrast of control sample.Any technology that detects gene structure or sequence change that is applicable to can be used to put into practice this method.For example, miRNA gene elmination or amplification can realize through the Southern Blot and the miRNA sequence-specific nucleic acid probe hybridization of genomic dna.Also can utilize sequential analysis and single strand conformation polymorphism.

The deletion of miRNA gene or amplification also can detect through these gene fragments of pcr amplification, and whether analyze amplification is come out from patient DNA sample fragment sequence or length through order-checking or electrophoresis identical with contrast.The deletion of miRNA gene also can be identified through near the deletion of the chromosomal marker it.

MiRNA gene level in a patient's the cell also can be assessed through the copy number of at least one miRNA gene in the measure sample; Wherein gene has only a copy number to mean that this patient has the possibility of suffering from the esophageal carcinoma; Rather than on the somatic chromosome with female chromosome on two copies, a copy on neither male sex's karyomit(e).

Any technology that detects gene copy number that is applicable to can be applied to this method, comprises Southern Blot and pcr amplification technology.The method that also has miRNA copy number in the definite human esophageal carcinoma sample is all closely to link to each other with chromosomal marker or other genes according to a lot of miRNA or gene cluster.Losing of a miRNA gene that links to each other with mark or other heterozygous geness can be learnt from the information of heterozygosity or marker gene among the patient.Therefore the technology of confirming the heterozygosity mark also can be used for this method.

" control sample " can be from the tissue sample that does not have patient with esophageal carcinoma.Or from the set of a group patient's tissue sample.

MiRNA 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35 among Fig. 1, or the gene level of 38 any or its homologues can be confirmed.When using more than the gene level of a miRNA but and inconsistently show that the indication of " great majority " can be considered to detected result when the esophageal carcinoma was arranged.For example, when using 5 miRNA, wherein 3 hints have the esophageal carcinoma, and the result can think that the hint this person is diagnosed as the esophageal carcinoma.Yet in some cases, the diagnosis of the esophageal carcinoma needs at least one or the more how special distinctive variation of miRNA.

Have multiple technologies can be used for confirming the miRNA gene level, the allele-specific that for example is included as on the array extends, and the PCR/LDR general array is based on single base chain extension of droplet, the sequencing by hybridization of sequence label molecular inversion probes and combination.

Esophageal carcinoma patient's sorting technique

On the other hand, this provides a sorting technique for the oesophagus patient, for example, and the classification of level of differentiation, tumour stage and pathology.Just having provided an oesophagus patient specifically (comprises such as the level of confirming differentiation; Confirm the tumour stage; Pathological classification with the oesophagus patient) sorting technique; Comprise the level of at least a miRNA of detection (for example miRNA in the table 2 or their homologue) or the miRNA gene level in the esophageal carcinoma patient tissue, be used as oesophagus patient classification's basis in the level (perhaps corresponding miRNA gene level) of this miRNA.

This provides a kind of method for confirming the level of differentiation in the patient with esophageal carcinoma; Comprise the level of at least a miRNA (or corresponding gene) in patient's human esophageal carcinoma among Fig. 2 a, be used as the basis of confirming esophageal carcinoma level of differentiation in this miRNA (or corresponding gene) level.For example, can confirm patient's high, medium and low level of differentiation through the miRNA level of measuring.Relation between miRNA (or corresponding gene) level and the level of differentiation also can confirm, for example, can realize through analyzing the level of miRNA (or corresponding gene) that is divided into the esophageal carcinoma colony sample of different level of differentiation with known method.

MiRNA can be hsa-miR-25; Any one or their homologue of hsa-130b and hsa-miR-130a; MiRNA level and level of differentiation in the esophageal carcinoma sample are opposite; That is to say that low-level miRNA is associated with high level of differentiation, and high-caliber miRNA is associated with low level of differentiation.Sometimes at least one miRNA is hsa-miR-335.Sometimes at least one miRNA is hsa-miR-25.Sometimes at least one miRNA is hsa-miR-130b.Sometimes at least one miRNA is hsa-miR-130a.Sometimes at least one miRNA is hsa-miR-181d.

This provides a kind of method for esophageal epithelial cell cancer patient pathological classification; Comprise the level of at least a miRNA (or corresponding gene) in patient's human esophageal carcinoma among Fig. 2 b, be used as the basis of classification of patient's pathology in this miRNA (or corresponding gene) level.For example, certain patient might be confirmed as fungate or medullary substance shape esophageal epithelial cell cancer, and this depends on detected miRNA level.Relation between the level of miRNA (or corresponding gene) pathological state different with cancer can be set up, and for example, can realize through analyzing the level that is divided into the miRNA (or corresponding gene) of the sample of different pathological classification with known method.

This provides a kind of method for confirming the tumour stage in the patient with esophageal carcinoma; Comprise the level of at least a miRNA (or corresponding gene) in patient's human esophageal carcinoma among Fig. 2 c, be used as the basis of confirming the tumour stage in this miRNA (or corresponding gene) level.For example, certain patient might be confirmed as I, II, or the tumour in III stage, and this depends on detected miRNA level.Sometimes certain patient has been determined T1, T2, or the tumour in T3 stage, and this also depends on detected miRNA level.Sometimes certain patient is determined the tumour in N0 or N1 stage, and this also depends on detected miRNA level.The level of miRNA (or corresponding gene) and the relation between the tumour different steps can be set up, and for example, can realize with the level that known method is divided into the miRNA (or corresponding gene) of the sample in different tumour stage through analysis.

Some systems (for example microarray) also are provided here; These systems have comprised and can (comprise Fig. 2 a by detection table 2; 2b, any miRNA among the 2c or its homologue) in probe and the decision table 2 of miRNA in the system of level of gene or its homologue of miRNA.These systems miRNA (or its corresponding gene) level and patient with esophageal carcinoma classified of great use in decision table 2.Therefore, at least a miRNA or system's (for example microarray) of its homologue (or corresponding gene) level that this provides in the definite table 2 have comprised most probes in this system, and each probe can detect a kind of miRNA (or its gene); At least 15% (comprises 20%, 30%, 40%; 50%, 60%, 70%; 80%, 90%, or 95%) probe can detect miRNA or its homologue (or corresponding gene) in the table 2.This provides a system (like microarray) for esophageal carcinoma patient classification, in this system, has comprised most probes, and each probe can detect a kind of miRNA (or its gene); At least 15% (comprises 20%, 30%, 40%; 50%, 60%, 70%; 80%, 90%, or 95%) probe can detect miRNA or its homologue (or corresponding gene) in the table 2.This provides a system (like microarray) for suffering from the carcinoma of esophagus patients classification, in this system, has comprised at least 2,5,10,20,30, or 40 probes, and each probe can detect a kind of miRNA or its homologue (or its corresponding gene) in the table 2.

Here the system that provides in addition can also be used to esophageal carcinoma patient is classified.For example, provide a useful system to classify to suffering from carcinoma of esophagus patients here, in this system, comprised one or more probes, each probe can detect miRNA or its homologue (or its corresponding gene) in a kind of table 2.Here provide a useful system can be used for confirming the level that the esophageal carcinoma is broken up, this system has comprised one or more probes, and each probe can detect miRNA or its homologue (or its corresponding gene) among a kind of table 2a.Here provide a useful system can be used for esophageal epithelial cell cancer patient is carried out pathological classification, this system has comprised one or more probes, and each probe can detect miRNA or its homologue (or its corresponding gene) among a kind of table 2b.Here provide a useful system can be used for confirming the tumour stage of patient with esophageal carcinoma, this system has comprised one or more probes, and each probe can detect miRNA or its homologue (or its corresponding gene) among a kind of table 2c.

Also the described probe that is used for detecting the system of miRNA (or its corresponding gene) is provided for making.Here for making one or more probes that the patient with esophageal carcinoma categorizing system is provided, each probe can be confirmed a miRNA or its homologue (or its corresponding gene) level in the table 2.Here for production provides a probe that is used for patient with esophageal carcinoma is carried out categorizing system (like microarray), each probe can detect the level of different miRNA (or its corresponding gene), and at least 15% (comprises 20%; 30%, 40%, 50%; 60%, 70%, 80%; 90%, or 95%) probe can detect a kind of miRNA or its homologue (or its corresponding gene) in the table 2.

This provides a kind of or multiprobe more for system's production of confirming esophageal carcinoma level of differentiation, and every kind of probe can be confirmed a miRNA or its homologue (or its corresponding gene) level among Fig. 2 a.This provides a kind of or multiprobe more for system's production of confirming esophageal epithelial cell cancer patient pathological classification, and every kind of probe can be confirmed a miRNA or its homologue (or its corresponding gene) level among Fig. 2 b.This provides a kind of or multiprobe more for system's production of confirming the esophageal carcinoma patient tumour stage, and every kind of probe can be confirmed a miRNA or its homologue (or its corresponding gene) level among Fig. 2 c.

The existence of the method for prognosis and composition and promotion patient with esophageal carcinoma

This invention for example comprises the method for confirming cancer of esophagi people living rate for esophageal carcinoma patient prediction provides method on the other hand.The Forecasting Methodology of this invention is being confirmed aspect esophageal carcinoma patient's the therapeutic process of great use.For example, confirm that the possibility of existence can help to determine that using conservative treatment still is radical therapy, or whether various modality can be used in combination.In addition, this prognosis can help to determine to improve whether the medicament (like the medicament of mentioning) of existence is necessary or ineffective here.

Here for patient with esophageal carcinoma prognosis survival probability method is provided; Comprise: (a) confirm the level of at least a miRNA in patient's the human esophageal carcinoma sample and (b) miRNA level and the threshold value of above-mentioned sample compared, wherein with threshold ratio miRNA level and patient survive be associated or anti-phase related." being associated " here refers to threshold value and compares the low survival rate of low-level miRNA hint, and vice versa." anti-phase related " here refers to be compared high-caliber miRNA with threshold value and hints high survival rate, and vice versa.

Sometimes, at least a miRNA is the miRNA that regulates goal gene, and goal gene is elected in one group of gene, comprising: PPP6C, SATB2, CHST11, CRELD1, ESRRA, MTMR4, RNF125; SYNJ1, TAF5, YWHAH, ZYX, CHST11, KIAA1033, TGFBR3, SNRK, RNF125, AXIN2, CAPZA2, SYNJ1, DLL1; YWHAH, MTMR4, PPP6C, CAMKV, and TAF5, PPP1CB, POU4F2, MYH1, MYH2, TOP2B, STX17, GBAS, MYH4; CPSF4, EIF4EBP3, LHX4, CLK3, CAPN6, KIAA1622, AUH, PPIF, KCNK3, IL6R, CSNK2A2, ZNF579, NRGN; CUL3, CIB2, ZBTB26, GPBP1, TMEM16D, HOXA1, CAMTA1, MCM3AP, MPPED2, HOOK2, PLAU, MCFD2, BLCAP; DHX15, FBN1, NCOA6, SNRPC, CCK, SFRS15, TMOD1, GPRC5B, ZNF403, DCUN1D5, ZNF423, and GPR64.Sometimes, at least a miRNA can regulate the goal gene that is selected from next group gene: PPP6C, SATB2, CHST11, CRELD1, ESRRA, MTMR4, RNF125; SYNJ1, TAF5, YWHAH, and ZYX..Sometimes, at least a miRNA can regulate the goal gene that is selected from next group gene: CHST11, KIAA1033, TGFBR3, SNRK, RNF125, AXIN2, CAPZA2, SYNJ1, DLL1, YWHAH, MTMR4, PPP6C, CAMKV, and TAF5.Sometimes, at least a miRNA can regulate the goal gene that is selected from next group gene: PPP1CB, POU4F2, MYH1, MYH2, TOP2B, STX17, GBAS; MYH4, CPSF4, EIF4EBP3, LHX4, CLK3, CAPN6, KIAA1622, AUH; PPIF, KCNK3, IL6R, CSNK2A2, ZNF579, NRGN, CUL3, CIB2; ZBTB26, GPBP1, TMEM16D, HOXA1, CAMTA1, MCM3AP, MPPED2, HOOK2; PLAU, MCFD2, BLCAP, DHX15, FBN1, NCOA6, SNRPC, CCK; SFRS15, TMOD1, GPRC5B, ZNF403, DCUN1D5, ZNF423 and GPR64. sometimes, the corresponding gene of miRNA is arranged on any of 5,10, No. 9 karyomit(e)s.Sometimes, miRNA is any or their homologue among Fig. 3.Sometimes, at least one miRNA is hsa-miR-103.Sometimes, at least one miRNA be has-miR-107. sometimes, at least one miRNA is has-miR-23b.

Here for patient with esophageal carcinoma prognosis survival probability method is provided; Comprise: (a) confirm the level of at least a miRNA in patient's the human esophageal carcinoma sample and (b) the miRNA level and the threshold value of above-mentioned sample compared; Wherein related with threshold ratio miRNA level and the above-mentioned patient anti-phase of surviving; This wherein at least one miRNA be hsa-miR-103, hsa-miR-107, or hsa-miR-23b or their corresponding homologue.Sometimes, at least one miRNA is hsa-miR-103.Sometimes, at least one miRNA is has-miR-107.Sometimes, at least one miRNA is has-miR-23b.

Here the miRNA level of narration also might reflect the variation (like miRNA described herein) of miRNA gene level.Sometimes; This is to suffer from the method that carcinoma of esophagus patients provides a prognosis; Comprise the level of analyzing at least a miRNA gene (as with hsa-miR-103; The miRNA gene of corresponding or its homologue of hsa-miR-107 and hsa-miR-23b), wherein the change with control sample icp gene level is hinting high or low survival rate.For example; Sometimes, this is to suffer from the method that carcinoma of esophagus patients provides a prognosis, comprises analyzing at least a and hsa-miR-103; Hsa-miR-107; With the miRNA gene of the corresponding amplification of hsa-miR-23b, relative comparison miRNA gene wherein, the miRNA gene of amplification is associated with low survival rate.Sometimes, this is to suffer from the method that carcinoma of esophagus patients provides a prognosis, comprise confirming at least a and hsa-miR-103, and the copy number of the corresponding miRNA gene of hsa-miR-107 and hsa-miR-23b, wherein copy number hangs down survival rate more than 2 hint.

A kind of method also is provided in addition, has promptly utilized the system to detect the probe of miRNA (or its corresponding gene) level or to contain one or more probes to come prognosis.For example; Sometimes; Utilize one or more probes system of one or more probes (or contain) to confirm the chances of survival of patient with esophageal carcinoma, the miRNA of probe wherein in can test sample compares with threshold value that its miRNA level is associated with above-mentioned patient's chances of survival or anti-phase is related.Sometimes; This provides one or more to be used for the probe into the patient with esophageal carcinoma prognosis, and it is related that the chances of survival of comparing miRNA level and above-mentioned patient with threshold value is anti-phase, and wherein at least a miRNA is hsa-miR-103; Hsa-miR-107, or hsa-miR-23b or their corresponding homologue.

Here one or more probes are provided for the manufacturing of reagent (or system); This reagent (or system) can be used for to suffering from the carcinoma of esophagus patients prognosis; The miRNA level of these probes in can test sample compares with threshold value that its miRNA level is associated with above-mentioned patient's chances of survival or anti-phase is related.Sometimes; One or more probes are provided here; Can be used for to suffering from the carcinoma of esophagus patients prognosis, the chances of survival anti-phase of comparing its miRNA level and above-mentioned patient with threshold value is related, and wherein at least a miRNA is hsa-miR-103; Hsa-miR-107, or hsa-miR-23b or their corresponding homologue.

This invention also provides method for the people who suffers from the esophageal carcinoma improves living state.Specifically, comprise that taking the effective dose of medicine thing to the patient lowers the miRNA level, compare that the level of this miRNA and above-mentioned patient's chances of survival are inverse correlation with threshold value.Here for medicament manufacturers provides a kind of medicament, improve the survival state of suffering from carcinoma of esophagus patients thereby use this medicament can reduce the miRNA level, to compare with threshold value, it is related that this miRNA level and above-mentioned patient's chances of survival are anti-phase.

This provides a method for suffering from the carcinoma of esophagus patients situation of making the life better; Comprise that taking the effective dose of medicine thing to the patient lowers the miRNA level; This miRNA is selected from and comprises hsa-miR-103, hsa-miR-107, or one group of miRNA of hsa-miR-23b or their corresponding homologue.Here the medicament that provides a kind of improvement to suffer from the survival state of carcinoma of esophagus patients for medicament manufacturers, this medicament can reduce the level of above-mentioned miRNA.

Method in this narration possibly at first be thought of as prognosis of patients (for example with the method for mentioning) here, considers giving of medicament again.

Sometimes, the level more than a miRNA has decline.This can realize through utilizing the medicament that for example lowers two or more miRNA levels.Also can use two or more medicaments to lower the level of two or more miRNA.For example; Sometimes; This provides a method for suffering from the carcinoma of esophagus patients situation of making the life better, and comprises that one or more medicines of taking effective dose to the patient lower at least two kinds of miRNA levels, and these miRNA are selected from and comprise hsa-miR-103; Hsa-miR-107, or one group of miRNA of hsa-miR-23b or their corresponding homologue.Sometimes; Here for providing one or more improvement, medicament manufacturers suffers from the medicament of the survival state of carcinoma of esophagus patients; This medicament can reduce the level of at least two kinds of miRNA; These miRNA are selected from and comprise hsa-miR-103, hsa-miR-107, or one group of miRNA of hsa-miR-23b or their corresponding homologue.Sometimes, this provides a method for suffering from the carcinoma of esophagus patients situation of making the life better, and comprises that one or more medicines of taking effective dose to the patient lower hsa-miR-103, hsa-miR-107, or the level of hsa-miR-23b.Sometimes, suffer from the medicament of the survival state of carcinoma of esophagus patients for medicament manufacturers provides one or more improvement here, this medicament can reduce hsa-miR-103, hsa-miR-107, or the level of hsa-miR-23b.

Acceptable carrier on a kind of drug component and a kind of pharmaceutics of the miRNA of reduction level is provided here, and wherein at least a miRNA is hsa-miR-103, hsa-miR-107, or hsa-miR-23b.Sometimes, at least a miRNA is hsa-miR-103.Sometimes, at least a miRNA is hsa-miR-107.Sometimes, at least a miRNA is hsa-miR-23b.Sometimes, this medicament is double-stranded RNA (for example short or siRNA or " siRNA "), antisense nucleic acid chain, or RNA molecule such as ribozyme with enzymic activity.The method of the situation of making the life better will be explained below.

Here survival state that said survival state can be no disease or comprehensive survival state.At this, " DFS situation " is meant and tumor recurrence and/or the individual destiny of diffusion and diagnosis back do not occur, and for example, someone hereafter tumour is not recurred." total survival state " is meant the destiny behind the patient diagnosis, and no matter whether tumor recurrence.

The existence prognosis

Some are for survival on the basis of method and the miRNA level that method described here is based on relative threshold of person's prognosis.

Threshold value can decide through several different methods, and given threshold can provide a boundary line, and patient's survival rate that should exist one group of miRNA level to be higher than threshold value is different with patient's survival rate that another group miRNA level is lower than threshold value.

Threshold value can be confirmed through the miRNA level in the human esophageal carcinoma sample of non-cancer.Also can decide through the miRNA level that analysis suffers from the crowd of the esophageal carcinoma.This can come perfect through histogram analysis, and the people's that a group is to be detected data display comes out among the figure, and wherein an axle is represented the miRNA level, the survival rate that another expression is individual.Two or more independently colonies can be divided into different subgroups through identical or different miRNA level and confirm.For example, in some cases, threshold value can basis based on the M.L. of the people's of the people of a group high viability and the low survival rate of a group miRNA on.Threshold value also can be represented two or more miRNA.This can embody through the ratio of every kind of miRNA level.

Threshold value can be an individual digit, can apply to each patient with esophageal carcinoma, or different and different according to concrete subgroup.For example, aging man maybe be different with young man's threshold value, and the woman maybe be different with man's threshold value.Further, a threshold value can be that a people confirms.For example, threshold value possibly be a definite ratio, the ratio of the miRNA level of the relative non-cancer tissue of promptly same philtrum human esophageal carcinoma.

Threshold value can will be lower than threshold value and distinguish with the cancer of esophagi people living probability that is higher than threshold value, and this can realize through single argument or multivariate analysis.These methods are confirmed possibly concern between one or more variablees and the given result.Under special circumstances, these methods can confirm that a miRNA level and cancer patients do not have possibly get in touch between disease or the comprehensive survival state.Any method that in this field, can be used for carrying out this alanysis for people knew can be utilized.The example of univariate analysis such as Kaplan-Meir method or the Cox proportional-hazards regression model.

For example confirm threshold value, can confirm that with the patient of a group sufficient amount two or more sets independently have the patient of different levels miRNA based on the crowd through histogram.Typically, the group comprises at least 25 patients like this, comprises at least 50,75,100,125,150, or 200.Similarly, confirm that threshold value can comprise at least 25 patients, comprise at least 50,75,100,125,150, or 200.

Further, a threshold value can be separated two groups of patients, possibly exist a plurality of threshold values can separate a large amount of patients.For example, the patient that two threshold values can at first be separated one group of high level miRNA separates the patient of one group of medium level then, remaining one group of low-level patient.The different threshold values of some amount can be described a curve, and for example a successive line can be described the possibility that patient does not have survival state disease or comprehensive.This curve constitutes the miRNA level of " continuously ", and patient does not have disease existence or the possibility and the interior miRNA level of its body of survival state is proportional comprehensively.Two or more miRNA levels curve thus represent.

In some cases, between this is for miRNA in the invention of cancer patient prognosis, possibly make up each other.The prognosis decision that is combined as of two or more miRNA has played more important role.

The miRNA level also can be used for combining with its dependent variable; This variable possibly be significant on statistics, hint esophageal carcinoma patient's the no disease or the comprehensive possibility of survival state, for example pathology hint (age for example; The tumour size; Tumor histology, clinical stage, family history or the like).For example, the clinical stage of cancer has the no disease of hint or the comprehensive significance of existence on statistics, this threshold value maybe basis in the different clinical stage and difference.Therefore, the threshold value of miRNA maybe be according to the difference of another indication parameter and difference.

In an exemplary method, Kaplan-Meier analyzes and is used for confirming the relation between survival rate and the miRNA level.

In some cases; This method comprises: (a) detect in the individuality level of at least a miRNA in the human esophageal carcinoma, (b) individuality be divided into first group and second group, wherein first group to have a low-level miRNA possible with bigger existence; Second group has high-caliber miRNA and less existence possibility; At this at least a miRNA is hsa-miR-103, hsa-miR-107, or hsa-miR-23b.

After confirming the level of one or more miRNA and comparing, just can patient be assigned to the group of the corresponding no disease or the possibility of comprehensively surviving with threshold value.This patient possibility of not having disease or comprehensively existence is confirmed with regard to the possibility that has which lineup not have disease or existence comprehensively then.

For example, a sample can be considered to have low-level miRNA.This patient is then assigned to the patient group of low-level miRNA.Because confirmed that the patient of high-level miRNA has the possibility of higher no disease or comprehensive survival state, so particular patient may be considered to have the possibility of higher no disease or comprehensive survival state.

Method described here can further be that the individual confirms suitable therapeutic process.One of them method commonly used is exactly to be the cancer patient prognosis in this field, and cancer is early stage and Advanced Carcinoma Patient is different.For example, for cancer patients's prognosis in I stage possibly tended to cancer continued growth or transfer, then possibly tend to effective cancer treatment method to the cancer patients in IV stage.Suitable therapeutic modality confirm will consider these parameters.

Improve the method and the measure of living state

The method of improving cancer patients's survival state also is provided here, and this realizes miRNA for example shown in Figure 3 through using the medicament that reduces specific miRNA level.

Any medicament that can reduce the miRNA level may be used to the method for this invention.The medicament of suitable inhibition miRNA genetic expression comprises double-stranded RNA (for example short or siRNA or title " siRNA "), antisense nucleic acid, RNA molecule such as ribozyme with enzymic activity, small molecules mixture and protein.These medicines can use separately also and can be used in combination (other drug of for example mentioning) here.These medicines are (as suppressing expression or the function of miRNA) or the indirect level that reduces miRNA directly (as through influencing the level of miRNA gene).

For example, the expression of a given miRNA can be passed through double-stranded RNA (dsRNA) inductive RNA and disturb inhibition, and this double stranded rna molecule and miRNA gene product have at least 70%, comprise at least 75%; 80%, 85%, 90%, 95%; 98%, 99%, or 100% sequence homology.In some cases, double-stranded RNA is exactly " short or siRNA " or claim " siRNA ".

The siRNA that in current approach, uses is made up of the double-stranded RNA of 10-30bp, comprises for example 12-28,14-26,16-24, or 18-22 any one.SiRNA comprises a RNA chain that an adopted RNA chain and a reverse complemental are arranged, and the two combines through the Watson-Crick basepairing rule.Sense strand comprise one section with target miRNA in the sequence of nucleic acid array complementation.Have justice and the antisense strand of siRNA can comprise two complementary, single stranded RNA molecule, or only comprise a single molecule, and its complementary portion has a chain to amount to valency through base pairing to be connected to form " hair fastener " structure and to form.

Insertion through single or a plurality of bases, disappearance, replacement, variation etc., siRNA is different with natural RNA.This variation comprises the insertion of non-nucleosides material, for example in the terminal of siRNA or one, two nucleosides, or siRNA resist the modification of ribozyme digestion.In some cases, one of siRNA or two chains also comprise 3 ' protruding terminuses.SiRNA can obtain through chemosynthesis or biosynthesizing, perhaps expresses obtaining through recombinant plasmid or virus vector, and this will be explained below.

The expression of given miRNA can be suppressed by antisense nucleic acid." antisense nucleic acid " here refers to through RNA-RNA or RNA-DNA and interacts, and with target RNA bonded nucleic acid molecule, so just changed the activity of target RNA.The antisense nucleic acid that is applicable to present method is the sequence complementary single-chain nucleic acid (for example, RNA, DNA, RNA-DNA mosaic, PNA, and LNA) that adjoins with miRNA.In some cases, antisense nucleic acid is to have 70%, 75%, 80%, 85%, 90%, 95% at least, or 100% and miRNA adjoin sequence complementary nucleotide sequence.In some cases, antisense nucleic acid has 10-30 nucleosides, comprises for example 12-28,14-26,16-24, or 18-22 nucleosides.

Antisense nucleic acid also can have specific modification to strengthen target specificity, ribozyme resistibility, transportation or other characteristics relevant with efficient on nucleic acid backbone or sugar or base.These modifications comprise SUV, interact like acridine or comprise one or more ribozyme resistance groups.

Antisense nucleic acid can produce through chemical process or biological method, perhaps also can obtain from recombinant plasmid or expressing viral.This will be explained below.

The nucleic acid that the genetic expression of given miRNA also can be had enzymic activity suppresses.Here " nucleic acid with enzymic activity " refers to the nucleic acid that comprises the substrate calmodulin binding domain CaM, the nucleic acid array complementation that this zone and miRNA adjoin, cracking miRNA specifically.In some cases, the calmodulin binding domain CaM of the nucleic acid of enzymic activity and miRNA contiguous zone 50-100% are complementary, comprise like 75-100% or 95-100% complementary.The nucleic acid of enzymic activity also can include modification at base, glycosyl or phosphate.The nucleic acid with enzymic activity that typically is used for current method is exactly ribozyme.

Nucleic acid with enzymic activity can produce through chemical process or biological method, perhaps also can obtain from recombinant plasmid or expressing viral.This will be explained below.

With the nucleic acid molecule transfered cell, comprise cancer cells, method in this field, have a lot.These methods comprise microinjection, and electroporation is liposome-mediated, the conversion of calcium phosphate mediation; (macromolecular complex for example, pearl are transmitted in the conversion of DEAE-VISOSE mediation, microparticle bombardment, colloid scattering system; Fat liquor in the water, micella, mixed micelle and liposome); And and antibodies, gramacidinS, artificial virion or other carriers such as TAT.

Utilize suitable carriers also can nucleic acid drug be imported in the mammalian cell in vivo or in vitro.Suitable carriers comprises virus vector and non-virus carrier such as plasmid vector.This carrier is very useful to medicament that therapeutic dose is provided such as sense-rna or siRNA.

System based on virus has an advantage: can high-caliber relatively heterogeneous nucleic acid be imported in the different cells.The suitable virus vector of mediation nucleic acid comprises, herpes simplex virus carrier for example, vaccinia virus vector, cytomegalovirus carrier, murine leukemia virus carrier, adenovirus carrier, gland relevant viral vector, retroviral vector and lentiviral vectors.The orientation movement of virus vector also can be through modifying with other viral envelope proteins or surface antigen carrier.For example, the AAV carrier can be modified by the surface protein of rabies blister virus (VSV), Ebola virus, Mokola etc.

Nucleic acid or carrier in this invention can contain inducible promoter or enhanser arbitrarily, thereby can be through adding the expression of stimulation or molecular Control sense-rna or siRNA.But this inducible system comprises for example tsiklomitsin inducible system; Heavy metal inductive rhMT; Moulting hormone inductive insect steroid hormone or related steroid such as curtain multitude sterone, the mouse mammary tumour virus (MMTV) of steroid such as glucocorticosteroid and estrogen-induced and temperature change inductive heat-inducible promoter.

The dosage that a kind of medicament can reduce the miRNA level is effective dose.In some cases, medicament can reduce 10%, 20%, 30%, 40% of difference between miRNA level and the threshold value, or 50%.The typical dosage of medicine (like nucleic acid drug) comprises the 0.1-3000mg/kg body weight, the 10-2000mg/kg body weight, and the 50-1000mg/kg body weight, the 100-500mg/kg body weight, but be not limited to this scope.In some cases, the dosage of medicine (like nucleic acid drug arbitrarily) is approximately the 10-500mg/g tumour, for example 20-300mg/g tumour, 50-200mg/g tumour and 100-150mg/g tumour.

There is a kind of technology commonly used can confirm the appropriate dose that a human therapy uses at an easy rate in this field.The classical frequency of medicament comprises per at least three weeks once, and is whenever biweekly weekly, weekly twice, on every Wendesdays time, on every Thursdays time, on every Fridays time, on every Saturdays time or once a day, but be not limited to these.In some cases, the timed interval of each medication is less than a week, comprises 6,5,4,3,2,1 days.In some cases, each administration time is constant at interval.For example, can be every day, per two days, per three days, per four days, per five days, or weekly.In some cases, can be every day twice, every day three times or more.

Administration time can be very long, as from one month to 3 years.For example, dosed administration can extend to 2,3,4,5, and 6,7,8,9,10,11,12,18,24,30 and 36 months.In some cases, the administration arrangement can not stop.In some cases, a week can not be longer than in the interval of medication.

The synthetics of describing once more can pass through the conventional route administration, including, but not limited to intravenously, and intraperitoneal, intraocular, intra-arterial, in the lung, oral; In the vesicle, intramuscular, subcutaneous in the tracheae, through skin,, partial through pleura; Suck, through mucous membrane, skin, stomach, intraarticular is in the ventricle; Rectum, vagina is in the skull, in the urethra, in the liver, in the knurl.In some cases, systematically administration.Be administration partly in some cases.

Medicinal composition also is provided here, comprises acceptable carrier on a kind of composition and the pharmacopedics of the miRNA of attenuating level.In some cases, above miRNA is selected from hsa-miR-103, hsa-miR-107, hsa-miR-23b or their homologue.In some cases, at least a is hsa-miR-103.In some cases, at least a is hsa-miR-107.In some cases, at least a is hsa-miR-23b.In some cases, this composition is siRNA.In some cases, this composition is a sense-rna.In some cases, this composition is a ribozyme.

In some cases, pharmaceutical cpd is aseptic.In some cases, pharmaceutical cpd is apyrogenic.

The acceptable carrier has water on the suitable pharmacopedics, water damping fluid, conventional salt, 0.4% salt, 0.3% salt and mucinase.Pharmaceutical cpd also possibly comprise traditional pharmacology vehicle, and/or additive.Suitable pharmacology vehicle comprises stablizer, inhibitor, penetration degree regulator, damping fluid, pH regulator agent.Suitable additive comprises the biological suitable damping fluid of physiology; Additional chelant (like DTPA or DTPA bisamide) or calcium chelated complexes are (like DTPA; The CaNaDTPA bisamide) or calcium or sodium salt (calcium chloride, calcium ascorbate, calglucon or lactic acid ca).The pharmaceutical cpd of this invention can be packed or freeze-drying with liquid form.

The solid pharmaceutical composition of this invention can be used acceptable non-toxic carrier on traditional pharmacopedics, for example, and the N.F,USP MANNITOL of pharmacy grade, lactose, starch, Magnesium Stearate, soluble saccharin, talcum, Mierocrystalline cellulose, glucose, sucrose, magnesiumcarbonate or the like.

MiRNA level detection system and/or miRNA gene level detection system

This invention has the miRNA level of characteristic change that multiple systems is provided for detecting the cancer of esophagi philtrum, and the system of definite miRNA gene status also is provided.As stated, these systems can be used for multiple purpose, comprise esophagus cancer diagnosis, and esophageal carcinoma patient classifies and is esophageal carcinoma patient prognosis.

System described herein comprises the probe that detects miRNA and/or miRNA gene state.Below discussion concentrate on the system that can detect miRNA, the people with the routine techniques this field in is readily appreciated that particular aspects described herein also is applicable to comprise and can detects genetically deficient, the system of the probe of amplification or the change of miRNA gene copy number.

For example, in some cases, a system that comprises most probes is provided here, each probe can test sample in different miRNA; Wherein at least 15% (comprises 20%, 30%, 40%, 50%; 60%, 70%, 80%; 90%, or 95%) probe can detect Fig. 1, the miRNA among table 2 or Fig. 3 or its homologue.In some cases, this system comprises at least 2,5, and 10,20,30,40, or 50 kinds of probes, wherein every kind of probe can detect Fig. 1, a kind of miRNA among table 2 or Fig. 3 or its corresponding homologue.

In some cases, a system that comprises most probes is provided here, wherein the miRNA of each probe in can test sample; Wherein at least 15% (comprises 20%, 30%, 40%; 50%, 60%, 70%; 80%, 90%, or 95%) probe can detect miRNA or its homologue among Fig. 1.In some cases, this system comprises at least 2,5, and 10,20,30,40, or 50 kinds of probes, wherein every kind of probe can detect a kind of miRNA or its corresponding homologue among Fig. 1.

In the application, provide the system that multiple probe is formed, wherein each probe can test sample in a different miRNA, at least 15% (comprises at least 0%; 30%, 40%, 50%, 60%; 70%, 80%, 90%; Or 95%) probe can detect a miRNA in the table 2 (comprising Fig. 2 a, any miRNA among 2b and the 2c).In the application, this system has 2,5,10,20,30 at least, or 40 probes compositions, and wherein each probe can detect miRNA or their homologue (comprising Fig. 2 a, any miRNA among 2b and the 2c) in the table 2.

In the practical application, provide the system that multiple probe is formed, wherein different miRNA in each probe ability test sample; At least 15% (comprises at least 0%, 30%, 40%; 50%, 60%, 70%; 80%, 90%, or 95%) probe can detect a miRNA or their homologue among Fig. 3.In the practical application, this system has 1,2,3,6,9,12,15,18 at least, and 21 probes of or are formed, and wherein each probe can detect the miRNA among Fig. 3.

In the system described herein with the identical miRNA of plural probe in detecting.For example, in practical application, when system is a chip, probe exists with multiple copy (as 2,3,4,5,6,7 or more).In the application, different probe detects identical siRNA in the system.For example, these probes are attached to the different zones (overlapping or non-overlapped) of miRNA.

In some instances, probe is an oligonucleotide.In order to detect miRNA, certain sequence variations is an acceptable.Like this, the sequence of oligonucleotide (perhaps his complementary sequence) and miRNA can deposit a little different.The professional and technical personnel knows, this sequence variations can not the remarkably influenced oligonucleotide be confirmed the ability of miRNA level.For example, the homologue and the varient of oligonucleotide are compared with standard method, confirm high relatively sequence similarity.Oligonucleotide in the invention and miRNA sequence have the similarity of 40% (comprising 50%, 60%, 70%, 80%, 90%, 95% or more) at least.In the application, the part oligonucleotide is used to detect miRNA and other albumen, and other parts are used for oligonucleotide and combine with matrix.In the application, other parts are made up of non-distinguished sequence (like polyT), increase the distance of complementary sequence part and stromal surface.

Oligonucleotide in the system comprises DNA, RNA, PNA, LNA, above combination or modified forms.They comprise that also the oligonucleotide that adopts modification is as main body.In some instances, oligonucleotide is at least by 9,10, and 12,13,14,15,16,17,18,19,20 or the more oligonucleotide composition complementary or identical with miRNA.One oligonucleotide is made up of plural complementary sequence.In some instances, active ingredient (like amino) combines with 5 ' or 3 ' end of oligonucleotide, makes it to combine with matrix.

In some instances, system is the micro-array chip that is furnished with probe.Micro-array chip and chip can exchange use, and its surface is an array, a regular array, and the supposition of the uncertain biological sample of the character that distributing combines (hybridization) site.In some practical applications, chip is the set that is fixed on the different oligonucleotides on the matrix specific position.

For example, in the application, the chip of being made up of multiple probe is provided, wherein different miRNA in each probe ability test sample; At least 15% (comprises at least 0%, 30%, 40%, 50%; 60%, 70%, 80%, 90%; Or 95%) probe can detect Fig. 1, table 2, or a miRNA and their homologue among Fig. 3.In the application, the chip of being made up of multiple probe is provided, wherein different miRNA in each probe ability test sample; At least 15% (comprises at least 0%, 30%, 40%; 50%, 60%, 70%; 80%, 90%, or 95%) probe can detect a miRNA and their homologue among Fig. 1.In the application, the chip of being made up of multiple probe is provided, wherein each probe can test sample in a different miRNA, at least 15% (comprises at least 0%; 30%, 40%, 50%; 60%, 70%, 80%; 90%, or 95%) probe can detect Fig. 1, a miRNA and their homologue in the table 2.In the application, the chip of being made up of multiple probe is provided, wherein different miRNA in each probe ability test sample; At least 15% (comprises at least 0%, 30%, 40%; 50%, 60%, 70%; 80%, 90%, or 95%) probe can detect a miRNA and their homologue among Fig. 3.

The micro-array chip of definite miRNAs gene state is provided.Micro-array chip is a classical way of confirming the gene state.For example, the molecular system of sequence label is used for confirming the gene state.

Chip can be processed on the matrix of multiple material, like paper, and glass, plastics (Vestolen PP 7052, nylon; PS), SEPIGEL 305, nitrocotton, silicon; Optical fiber or other solid-state or semi-solid state upholders that is fit to form plane conformation (like sheet glass, silicon chip) or three-dimensional conformation (pin type, fiber; Pearl, particle, microwell plate, kapillary).

In practical application, probe is an oligonucleotide, and the oligonucleotide of compositing chip can combine with matrix through several different methods, is not limited to, and the in situ hybridization (for example high-density oligonucleotide chip) of optical lithography is adopted in (1); (2) at glass, point or India and China's low density chip on nylon or the nitric acid film; (3) adopt concealment; (4) on nylon or the plain Hybond membrane of nitric acid, put the marking.Oligonucleotide utilization hybridization, magnetic bead, perhaps fluid phases such as micropore dish or kapillary are through being fixed on the matrix of non covalent bond.

With nucleic acid and the classical technology of solid substrate (like sheet glass) bonded.A kind of method is base or the analogue that combines modification, and their part can be incorporated into solid substrate, and is for example amino, aminoderivative or other positively charged groups.The product of amplification with encapsulated solid substrate by aldehyde radical or other reactive groups and contact, like sheet glass, the product of amplification and reactive group formation covalent linkage, and combine with slide with the form of covalency.(CA) point sample instrument on the glass plate that aldehyde radical encapsulates, is made into chip with amplified production point for BioDot, Inc.Irvine to use Biodot.According to the step of delivering, with amplified production point at aldehyde slide (Schena et al., Proc.Natl.Acad.Sci.U.S.A. (1995) 93:10614-10619)..Chip also robotic arm is imprinted on glass, nylon (Ramsay, G., Nature Biotechnol. (1998); 16:40-44), Vestolen PP 7052 (Matson, et al., Ahal Biochem. (1995); 224 (1): 110-6), (Marshall, A.and Hodgson on the silicone resin sheet; J., Nature Biotechnol. (1998), 16:27-31).The method of other chip equipment comprise fine micro puncture in the electric field (Marshall and Hodgson, supra) with the direct point sample of polynucleotide on the flat board of positive electricity pocket cup.Certain methods is also used always, as www.cmt.corning.com with Http:// cmgm.stanford.edu/pbrown/Disclosed, carry out the method for chemical surface treatment with aminopropane silicone oil.

Prepare chip through preparation high-density nucleosides chip.As everyone knows, and the fast deposition of this technology employing polynucleotide (Blanchard et al., Biosensors & Bioelectronics, 11:687-690).Other prepare the method for chip, as through cover (Maskos and Southern, Nucleic.Acids.Res. (1992), 20:1679-1684).In principle, above any chip of being mentioned, can on the nylon Hybond membrane, use by dot blotting.But the chip that the professional and technical personnel is often first-selected very little is because they have less hybridization volume.

Test kit

This patent is that several different methods described herein provides test kit.

For example, in application, a test kit is provided, has comprised an individual system (like micro-array chip) that is used to detect the miRNA level.In application, test kit comprises the extra reagent that detects, and also furnish an explanation book or user manual are introduced the best practice of using this invention in detail, and these explanations are instructed and also can be obtained from the Internet.

In application, test kit provides a system (chip) that is used for diagnosing esophageal cancer.This test kit comprises that further control sample is used for confirming reference level, and/or obtains the information of reference level.In the application, test kit comprises specification sheets, introduces and how to use its diagnosing esophageal cancer.

In application, test kit provides the esophagus cancer patient's that is used to classify system (chip).It is individual that this test kit comprises that further control sample is used for classification, and/or the information of control sample.In the application, test kit comprises specification sheets, introduces and how to use its classification individual.

In the application, provide test kit to be used for confirming the prognosis of an esophagus cancer patient existence.For example, this test kit is made up of the probe that detects miRNA (as shown in Figure 3).In the application, test kit comprises that further control sample is used for confirming the information of critical level or relevant critical level.In the application, test kit also comprises specification sheets, instruct to use test kit to patient's prognosis of surviving.In the use, test kit comprises that also some reagent are used to reduce the miRNA level or the medicine synthetics is used to improve survival rate.

This test kit further comprises some reality, but is not limited to these, like substrate, and label, primer; The reagent of mark miRNA, the reagent of extracting miRNA is used to the feminine gender or the positive control of hybridizing and detecting, test tube and/or other annexes; The reagent of collection organization's sample, damping fluid, hybridizing box seals film etc.; Also comprise software package, as be used for the statistical method of miRNA horizontal analysis and/or the change of miRNA horizontal properties, select password and number of the account arbitrarily, be used to assess editor's DB.

In the application, comprise a pharmaceutical composition in the test kit, its composition can reduce the level of miRNA shown in Fig. 3, and how to use compsn to improve esophagus cancer patient's survival rate.In use, test kit comprises that carrier or other media are used to transport compsn.In the use, test kit also has the use of instructions direct pharmaceutical composition.

Following Example is used to illustrate this invention, but is not limited in this.

Embodiment 1

This example shows is analyzed the specimen preparation of miRNA level.

Patient and sample

Comprise 31 pairs former esophagus unicorn cancer cells and corresponding contiguous normal esophagus cancer tissue in the training set.These samples are gathered from the patient of tumour hospital of the Chinese Academy of Medical Sciences, and obtain informed consent.All tissue samples obtain through operation from the patient who never treated, and freezing rapidly in liquid nitrogen ,-80 ℃ of storages (being less than 6 years) are up to the miRNA extracting.Flesh tissue in second independent sets from 5 pairs of cases, as efficacy data collection independently, be to collect from identical hospital in 2006, the storage time is less than 6 months.Peripheral portion by the excision esophagus cancer encapsulates with paraffin, and section is dyeed with Chang GuiH &E (phenodin & eosin).The pathologist assesses tumour cell concentration and definite tumour] histology.The tracking data is from the trace data storehouse of Cancer Hospital of Chinese Academy of Medical Sciences.The clinical pathology information of all samples (age, sex, pathology, differentiation, TNM by stages, tumour stage, operation back survival time) all be available.This research is ratified by the medical ethics council of Cancer Hospital of Chinese Academy of Medical Sciences.

The preparation of miRNA chip

Have the miRNA chip design that 509 sophisticated miRNA sequences were assembled and were integrated into us; 435 people sources (miRNA that comprises 122 predictions that from deliver document, obtain) wherein are from 196 rats of miRNA DB and the sophisticated miRNA of 261 mouse.In addition, designed 8 short oligonucleotides, they and known RNA sequence be homology not; Adopt Ambion miRNA probe to make up test kit (Cat.No.1550; Austin TX), obtained the synthetic accordingly miRNA. of these 8 short oligonucleotides before analyzing through in-vitro transcription; These synthetic miRNA of different quantities are joined in people's the miRNA sample, as interior mark.

The ripe miRNA of the miRNA probe sequence of all designs and their homologous total lengths is complementary fully.For with probe-immobilized to aldehyde group modified surface of glass slide, these probe sequences couple together length has 40nt (the miRNA probe of 3 ' end adds the polyT of 5 ' end 19mer), and C6 5 '-amido modified is arranged.Oligonucleotide probe is synthetic at MWG Biotech, is dissolved in EasyArray ^TMIn the spotting solution, concentration is 40 μ M.Use SmartArray ^TMEach probe of microarrayer (CapitalBio Corp.) repeats point sample 3 times.

The mark of target RNA

With TRIZOL reagent extracted total RNA, low molecular weight RNA is used Ambion ' s miRNA Isolation Kit extracting.Use T4RNA ligase enzyme labeling technique according to Thomson ' protocol (Thomson et al., 2004).Briefly, (Dharmacon, Lafayette is CO) at T4RNA ligase enzyme (New England Biolabs, Beijing, China) mark under the effect by 5 ' of 500ng-phosphate-cytidyl-uridyl-cy3-3 ' for 4 μ g low molecular weight RNAs.Labeled reactant carried out 2 hours at 4 ℃.The RNA of mark uses washing with alcohol again with 0.3M sodium acetate and 2.5 times of volume ethanol depositions, is resuspended in after the drying in the 15 μ l hybridization buffers, contains 3 * SSC, 0.2% SDS and, 15% methane amide.

Slide hybridization

At LifterSlipTM (Erie; Portsmouth is hybridized in NH), and the hybridizing box method is at BioMixerTM (CapitalBio); So that the continual bulk crossing damping fluid of ability; Make the hybridization of whole slide more balanced, prevent fringing effect, the validity of this method is confirmed in our full genome mRNA expression chip.Hybridization is spent the night in 42 ℃ of water-baths.(at room temperature washed 5 minutes with 0.2%SSC with washing soln by 0.2% SDS, 2 * SSC) continuous washing 5 minutes at 42 ℃ for chip.Chip obtains image with the burnt LuxScanTM scanner scanning of copolymerization with LuxScanTM 3.0TM software analysis.

Computational analysis

The MV that repeats a little of every miRNA is removed background, and the laggard row of stdn is further analyzed.Adopt each chip intermediate value to carry out stdn, value matrix in the use.In all samples, from data, remove expression signal and be lower than 800 gene.Confirm the different miRNAs that express through chip significance analysis (SAM) (www-stat.stanford.edu/～tibs/SAM/index.html)).For each gene, the SAM basis provides a numerical value with respect to the expression changing conditions of the standard deviation of all measurements.Carry out hierarchical clustering according to average chain with Pearson's dependency.Support vector machine method is used for cross validation and prediction test set.

The available Algorithm Analysis target of miRNA is the most significantly disclosed with 4 kinds; Like MIRANDA (http://www.miRNA.org/); TARGETSCAN (http://www.targetscan.org/), PICTAR (http://pictar.bio.nyu.edu/) and miRBase ( Http:// miRNA.sanger.ac.uk/sequences/).In order to reduce false positive, the supposition target gene of being accepted will be predicted by 3 programs at least.The Kaplan-Meier curve be used for confirming miRNA express with from diagnosing to the dependency of treating the concluding time.

Table 1.

PCR in real time is analyzed

In order to verify the miRNA express spectra, extract full RNAs and carry out qRT-PCR with the specific primer of microRNA.ThermoScript II reaction conditions: the full RNA of 2.5ng/ μ l, 25nM root-ring RT primer, 1 * RT buffer, every kind of dNTPs 0.25mM, 200 U M-MLV ThermoScript II, 0.25U/ml RNase suppressor factor (Invitrogen).7.5ml reaction system temperature in MJ Research PTC-225 Thermocycler is bathed, kept 30 minutes at 16 ℃, 42 ℃ C30 minute, kept 5 minutes at 85 ℃, then 4 ℃ of preservations.All rts comprise the contrast of non-template, and a repetition is all arranged.PCR in real time carries out according to operational manual with FastStart DNA Master SYBR green I test kit and LightCycler.10 μ l PCR systems comprise 1 μ l RT product, 1 * PCR Master Mix, 15nM upstream primer and 15nM downstream primer.React 95 ℃ and kept 10 minutes, then carry out 40 circulations: 95 ℃ kept 15 seconds, and 60 ℃ kept 35 seconds, and 72 ℃ kept 3 seconds.All qRT-PCR reactions comprise the contrast of non-template, and a repetition is all arranged.Relative expression's ratio of miRNAs is confirmed in the point of crossing (CP) of employing cycle number.The gene expression analysis of people U6 is used as interior mark.The result analyzes with LightCycler software version 3.5 (Roche Diagnostics).Analyze the PCR in real time amplified production with solubility curve, and confirm with gel electrophoresis.Table 1 has been listed primer sequence.

Table 1.

Embodiment 2

The miRNA that this example shows changes in the esophagus cancer tissue sample expresses

More several expression to miRNA in the sample comprise bacterium shape and marrow shape, the classification in different tumour stages and the whole paired samples of squamous cell carcinoma.We have compared the expression intensity of 191 miRNA in the freezing sample at first.All cancers and other cancer sample chainly compare with Pearson's dependency through average, per sample between the similarity expressed carry out cluster.Initial clustering successfully is divided into two groups with 62 cancerous tissues and other cancerous tissue, except 1 cancer sample and 5 other cancer samples.

Compare each cancerous tissue and its corresponding non-cancerous tissue with the SAM method, have 40 miRNA in two groups of express spectras, to have statistical discrepancy.Like Fig. 1 and 4.Cancer sample through more different phenotypes and tissue typing obtains express spectra, compares each cancer sample and its corresponding non-cancer sample at last.We have confirmed to express different miRNAs in the different tissues classification with the cancer different steps.Adopt two kinds of analytical procedures, a kind of direct strength of signal, second kind of signal ratio according to cancer and other cancerous tissue according to cancerous tissue.The method of direct signal strength ratio ratio is confirmed more miRNA genes, and some genes all are significant in two kinds of methods, 5 gene (hsa-miR-335;-181d;-25 ,-7 ,-495) for pathological classification (bacterium shape vs marrow shape); Two genes (hsa-miR-25 ,-130b) for confirming the position of cancer at esophagus (go up among the vs under the vs).

The comparative analysis of the clinical esophagus cancer classification of table 2.

As independent sets, the miRNA express spectra of cold storage tissue acquisition is used in checking with fresh esophagus cancer tissue.Relatively, as shown in Figure 5 between the miRNA expression chip of 5 pairs of flesh tissues (test set) and the 31 pairs of cold storage tissues (training set).10 test sets are used the SVM method validation, and 8/10 specimen is by correct classification.Compare two detected miRNA of cancerous tissue, cold storage tissue detection to 191 miRNA, flesh tissue detect 164.155 miRNA occur in two set of organizations.In addition, 40 miRNA of the discovery of significance have different expression in the cancerous tissue of training set and test set and other cancerous tissue, explain that refrigerated esophageal tissue is keeping representing the miRNA level and the ratio of flesh tissue.

Embodiment 3

The relation of this example shows miRNA level and esophagus cancer patient existence.

Initial training set has 31 patient samples, is shorter than or is longer than 20 months according to existence after the initial diagnosis, is divided into two groups.Detect the intensity of each individual different miRNA that express according to the average signal of each existence group.The minimum detectable signal intensity that the average of these two average intensity value is analyzed as Kaplan-Meier.3 miRNAs (hsa-miR-103 ,-107 ,-low expression 23b) and high survival time (greater than back 90 months of diagnosis) have the dependency of height.At Fig. 3 and Fig. 6.The common function of most of miRNAs is reduced their target mRNA exactly and is translated as corresponding proteins matter, and the function of this dependency explanation mRNA is as tumor inhibitor.Ironically, the expression level of hsa-miR-23b is also confirmed among the I-III in neoplasm staging, but is only adopted direct signal intensity (table 2).Kaplan-Meier has analyzed the patient of DFS, has also confirmed 2 miRNAs (hsa-miR-103 ,-107), and low expression is relevant with the high DFS phase.

With PCR in real time analysis verification chip data.PCR in real time and chip all are used for hsa-miR-23b, and hsa-miR-103 and hsa-miR-107 confirm ripe miRNA abundance, as the signal to the esophagus cancer prognosis of patients.

Though aforesaid invention is described in detail, clearly understand purpose through chart and example, obviously for the technical professional can be skilled carry out less change and correction.Therefore, description and instance can not be interpreted as the limited use range of this invention.

Claims (3)

1. The probe is used in the preparation for determining the survival and prognosis of patients with esophageal cancer, and the probe is a probe that is completely complementary to the following minRNA respectively: hsa-miR-103:AGCAGCAUUGUACAGGGCUAUGA; hsa-miR-107:AGCAGCAUUGUACAGGGCUAUCA. 2. The application of the reagent in the manufacture of a pharmaceutical preparation that improves the survival of patients with esophageal cancer, characterized in that the reagent is a probe that is completely complementary to the following minRNA respectively: hsa-miR-103:AGCAGCAUUGUACAGGGCUAUGA; hsa-miR-107:AGCAGCAUUGUACAGGGCUAUCA. 3. A pharmaceutical composition, characterized in that the pharmaceutical composition is a probe fully complementary to the following minRNA respectively: hsa-miR-103:AGCAGCAUUGUACAGGGCUAUGA; hsa-miR-107:AGCAGCAUUGUACAGGGCUAUCA.

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