CN101921759B - A serum/plasma miRNA marker associated with cervical cancer and its precancerous lesions and its application - Google Patents
A serum/plasma miRNA marker associated with cervical cancer and its precancerous lesions and its application Download PDFInfo
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Abstract
本发明属于基因工程及肿瘤学领域，公开了一种与宫颈癌及其癌前病变相关的血清/血浆miRNA标志物及其应用。该标志物为miR-21单独或者miR-21与miR-29a的组合。该标志物及其引物可用于制备诊断试剂盒，用于宫颈癌及其癌前病变的辅助早期诊断。
The invention belongs to the fields of genetic engineering and oncology, and discloses a serum/plasma miRNA marker related to cervical cancer and its precancerous lesions and an application thereof. The marker is miR-21 alone or a combination of miR-21 and miR-29a. The marker and its primers can be used to prepare a diagnostic kit for auxiliary early diagnosis of cervical cancer and its precancerous lesions.
Description
Technical field
The invention belongs to genetically engineered and oncology, relate to a kind of serum/plasma miRNA marker and the application thereof relevant with cervical cancer and precancerous lesion thereof.
Background technology
Cervical cancer is one of modal malignant tumour of women, accounts for the second of global women's malignant tumour, is only second to mammary cancer.The whole world in 2002 is totally 49 ten thousand routine cervical cancer new cases, and wherein 83% case occurs in developing country, and 270,000 people are dead because of cervical cancer.Although the mortality ratio of China's city cervical cancer is on a declining curve after the sixties in last century, the sickness rate of cervical cancer and with and remain high in the mortality ratio of Rural areas always.Especially enter into the later stage nineties, the sickness rate of cervical cancer is the trend of obvious rejuvenation, urban middle-aged age at the sickness rate of the woman uterus cancer of 35-44 just with annual 4.1% speed increment.Chinese tumour registration statistical figure in 2005 show that China's cervical cancer pathogenesis rate is 9.1/10 ten thousand, and mortality ratio is 2.4/10 ten thousand.Cervical cancer has become one of important diseases that threatens China women life and health, is the great public health problem that needs to be resolved hurrily.
Cervical cancer be a slowly process, precancerous stage about 10-20 before occuring, human invasive cervix neoplasms is arranged usually, this infiltration last stage is known as cervical intraepithelial neoplasia (CIN) (cervical intraepithelial neoplasia, CIN) in the criteria for classification of WHO.Account for the thickness of epithelium of cervix uteri holostrome according to atypical cell, can further divide CIN can be divided into for 3 phases CIN, be slight intraepithelial neoplasia (CIN I), moderate intraepithelial neoplasia (CIN II), with severe intraepithelial neoplasia (CIN III), and carcinoma in situ ranges the CIN III phase.Develop into infiltrating carcinoma from the CIN I phase and need approximately time about 8-15, most of CINI is of short duration, nearly about 60% CINI is also reversible to be transferred to normally, and moderate and height CIN progress are very large for the possibility of cancer, has approximately can make progress above 10% CINIII to be infiltrating carcinoma.If but can hold well precancerous lesion during this period of time, and make in early days accurately diagnosis, in time take appropriate measures, just can greatly improve curative ratio and the survival rate of cervical cancer, even the generation of prevention cervical cancer.Therefore, diagnostic means seems particularly important effectively accurately.
At present, the diagnostic mode of cervical cancer mainly contains the inspection of uterine cervix Scrape method, vaginoscopy, uterine neck biopsy etc.Early stage and advanced cervical cancer has certain value although these methods are in diagnosis, because its specific degree and sensitivity are not high, traumatic larger, relate to again the aspect problems (such as misunderstanding, ignorance or the refusal of young woman for gynecologial examination) such as Social Culture ethics, not only can not guarantee to diagnose the accuracy to cancer patients's diagnosis, diagnosis capability for patient, especially young patient before the cancer that is in the slight CIN stage is more limited.In addition, for camber CIN patient, judge in early days which can make progress and be cervical cancer which then can be in steady state, the wound that also can avoid over-treatment to bring for a long time.Therefore, we need more clearly and effectively biomarker of discovery badly, cervical cancer and precancerous lesion thereof are made clear and definite auxiliary early diagnosis, and this will help the patient who is in precancerous lesion is in time taken appropriate measures, and precancerous lesion be reversed be normal circumstances; Simultaneously can help cervical cancer patient to make auxiliary early diagnosis, help early treatment, improve curative ratio and survival rate.
MicroRNAs (being miRNAs) is a focus of oncomolecularbiology research field in recent years, and its maturity state is the little single stranded RNA molecule that a class is about 19-23 Nucleotide, has high conservative in the evolution.It transcribes generation by DNA, but does not translate into protein, but regulates the function of other genes in protein building-up process.MiRNAs has participated in nearly all pathology and the physiological activity of Mammals, such as ontogeny, tissue differentiation, apoptosis and energy metabolism etc., exists closely and contacts with the generation of numerous disease, development.Since participating in lin-4 that regulation and control nematode sequential grows and being found with let-7, miRNA was selected in respectively the annual ten large technological breakthroughs of Science magazine twice at 2002 and 2003.Prediction miRNAs can regulate and control 5300 Human genomes, namely 30% of all genes at least in 2005.Along with going deep into of research, increasing miRNAs constantly is found.In recent years, the relation of miRNA and tumour has become the focus and emphasis of research, has been found that miRNA passes through expression and the lung cancer of negative regulator gene, mammary cancer, cancer of the stomach, the morbidity height correlation of cervical cancer etc.
Research has confirmed to exist in the serum/plasma miRNAs of hundreds of kind, these microRNAs s stable in properties, rich content, is easy to detection by quantitative, and has disease specific.Existing proven technique, the technology that comprises quantitative and qualitative analysis miRNA molecule, show that utilizing serum miRNAs will more effective as the method for Molecular biomarkers than traditional differential protein molecule marking method, simultaneously this has also overcome the bottleneck that molecule marker runs into antibody preparation and quantitative analysis development.
Yet, the report that also is not used at present the aspects such as the auxiliary early diagnosis of cervical cancer about serum/plasma miRNA serum s, if can filter out the serum/plasma miRNA serum s of cervical cancer and precancerous lesion unconventionality expression thereof as biomarker, and develop the monitoring of corresponding progression of disease, auxiliary diagnostic box, will be once strong promotion to the diagnosis present situation of China's cervical cancer.
Summary of the invention
Primary and foremost purpose of the present invention is for above-mentioned technical problem, proposes a kind of serum/plasma miRNA marker relevant with cervical cancer and the auxiliary early diagnosis of precancerous lesion thereof.
Second purpose of the present invention provides the primer of above-mentioned serum/plasma miRNA marker.
The 3rd purpose of the present invention provides above-mentioned serum/plasma miRNA marker and the application of primer in the test kit for preparing the auxiliary early diagnosis of cervical cancer and precancerous lesion thereof, dynamic monitoring thereof.
The 4th purpose of the present invention provides the test kit for cervical cancer and the auxiliary early diagnosis of precancerous lesion thereof.
The contriver by separate and the research cervical cancer patient and with the healthy women control serum/blood plasma of its age-matched in miRNAs, separate simultaneously and CIN I/II/III phase patients serum/blood plasma that Study On Age is complementary in miRNAs, seek one group with the high specific of cervical cancer and precancerous lesion height correlation thereof and the miRNAs of susceptibility, and develop cervical cancer and the precancerous lesion thereof that to be convenient to clinical application and assist early diagnosis kit, for examination and the diagnosis of cervical cancer and precancerous lesion thereof provides Data support, for dynamic monitoring and the clinical therapeutic efficacy evaluation of precancerous lesions of uterine cervix progress provides Data support.
The objective of the invention is to realize by following technical proposal:
A kind of serum/plasma miRNA marker relevant with cervical cancer and precancerous lesion thereof, this mark are independent or miR-21 and miR-29a combination of miR-21.
The primer of described serum/plasma miRNA marker, these primers are:
The primer of miR-21 is SEQ ID No.3 and SEQ ID No.4; The primer of miR-29a is SEQ ID No.11 and SEQ ID No.12.
The application of described serum/plasma miRNA marker in preparation cervical cancer and the auxiliary early diagnosis kit of precancerous lesion thereof.
The application of the primer of described serum/plasma miRNA marker in preparation cervical cancer and the auxiliary early diagnosis kit of precancerous lesion thereof.
A kind of cervical cancer and precancerous lesion thereof are assisted early diagnosis kit, and this test kit is independent or miR-21 and miR-29a combination for detection of miR-21 in the serum/plasma miRNA serum.
Described diagnostic kit, this test kit contain the primer of following two kinds of serum/plasma miRNA markers combination: the primer of miR-21 is SEQ ID No.3 and SEQ ID No.4; The primer of miR-29a is SEQ ID No.11 and SEQ ID No.12.
Described diagnostic kit, this test kit can also comprise PCR reaction enzyme and reagent commonly used, such as reversed transcriptive enzyme, and damping fluid, dNTPs, MgCl
2, DEPC water and Taq enzyme etc.; Can also contain standard substance and/or reference substance.
Specifically, the technical scheme that the present invention deals with problems comprises: (1) sets up sample storehouse and the database of unified standard: (SOP) gathers standard compliant blood sample with Standard operation procedure SOP, demography data and clinical data that systematic collection is complete.(2) serum/plasma miRNA serum differential expression spectrum analysis: select cases of cervical cancer, with the healthy women contrast of cases of cervical cancer age-matched, detect its serum/plasma miRNA serum express spectra and content, analyze general character and the characteristic of serum/plasma miRNA serum between the contrast of cases of cervical cancer and healthy women, screening differential expression miRNAs.(3) screening disease-related serum/plasma miRNA serum s: the serum/plasma differential expression miRNAs that has screened is carried out quantitative analysis in CIN I/II/III phase case, determine the precancerous lesions of uterine cervix serum/plasma miRNA serum s that is correlated with, can indicate the dynamic change of precancerous lesions of uterine cervix development.(4) development of serum/plasma miRNA serum examination and diagnostic kit: according to cases of cervical cancer and healthy women contrast, the relevant serum/plasma miRNA serum exploitation of CIN I/II/III phase case miRNAs auxiliary diagnostic box, realize the purpose of cervical cancer and precancerous lesion early diagnosis and monitoring of diseases active development.
The inventor gathers standard compliant blood sample with Standard operation procedure SOP (SOP), (these data can be used for judging progression of disease for the demography data that systematic collection is complete, clinical data etc., and the factors such as staging, patient age of controlling are for the impact of morbidity), and adopted RT-PCR, Real-time PCR method, Solexa sequencing technologies etc.
The experimental technique of research mainly comprises following components specifically:
1. the selection of research sample
The cases of cervical cancer of (1) clarifying a diagnosis through pathology;
(2) perform the operation and chemicotherapy without crossing before the blood sampling, without chemicotherapy before the operation;
(3) healthy women with the case age-matched contrasts;
(4) age-matched through CIN I phase, II phase, III phase case that pathology are clarified a diagnosis.
This research adopts 105 routine standard compliant samples to study altogether.
2.Trizol reagent (Invitrogen, Carlsbad, CA) extracts the total RNA of serum/plasma, according to a conventional method operation.Usually can obtain～5 μ g RNA/50ml serum or blood plasma.
3.Solexa order-checking
(1) total RNA carries out PAGE electrophoresis recovery 17-27nt RNA molecule
(2) will link primer (adaptor prime, Solexa check order universal primer) enzyme be associated in 3 of small RNA molecular ' with 5 ' end
(3) carry out checking order after the RT-PCR reaction
(4) data analysis and processing
4.Real-time RT-PCR (qRT-PCR) method
(1) gets experimenter's the total RNA of serum/plasma, obtain the cDNA sample by the RNA reverse transcription reaction;
(2) design primer;
(3) carry out the PCR reaction;
(4) variation of the amount of miRNA in detection and comparison cases of cervical cancer and the healthy women control serum/plasma sample, the variation that detects and compare the amount of miRNA in the CIN I/II/III phase case serum/plasma sample.
5. diagnostic reagent box preparation method
The Solexa method determines to have in cases of cervical cancer and the healthy women contrast miRNA of copy difference, large by qRT-PCR technology screening expression amount and difference degree in the contrast of cases of cervical cancer and healthy women, and the trend of the differential expression one group serum/plasma miRNA serum consistent with progression of disease trend in CINI/II/III phase case is as the index of cervical cancer and the auxiliary early diagnosis of precancerous lesion thereof.The serum/plasma miRNA serum relevant with cervical cancer and precancerous lesion thereof progress that filters out at last forms auxiliary diagnostic box (miR-21 separately and the combination of miR-21 and miR-29a).Diagnostic kit comprises miR-21 separately and the reagent such as the combination primer of miR-21 and miR-29a and Taq enzyme, dNTP.
6. statistical analysis technique
Use x
2Check (being used for classified variable) or the difference that student t check (being used for the continuous variable) is compared demographic characteristics, types of organization, CIN by stages and the average expression level of miRNA distributes between the research object group.
We find have 2 kinds of miRNAs and cervical cancer pathogenesis situation to have in the research of 30 routine cases of cervical cancer and 30 routine healthy women contrasts remarkable related.The different expression levels of 2 kinds of miRNAs are with 2
-Δ CtExpression, wherein Δ Ct=CT
Sample-CT
Confidential reference items, we detect at each, and adding six parallel shared Healthy Peoples contrast sample as reference in the version, calculate relative expression quantity.Further detected to these 2 kinds of miRNAs in each 15 routine patient of CIN I/II/III phase, and then whether observation miR-21 combination independent and miR-21 and miR-29a plays an important role in the genesis of cervical cancer.
Statistical analysis all by special statistical analysis software finish (SAS, v.9.1.3).The horizontal P value of significance,statistical is made as 0.05, and all statistical test are two-tailed test.
Below be further instruction of the present invention:
In above-mentioned 30 routine qualified cases of cervical cancer and 30 routine healthy women contrasts, two groups of ages are by individual exact matching.We obtain correlated results with these two groups of crowds through Solexa order-checking test.
According to the Solexa sequence measurement, the inventor detects the miRNA that there are differences expression in the serum of " cases of cervical cancer " group and " healthy women contrast " group and comprises: let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, miR-1, miR-100, miR-101, miR-103, miR-106b, miR-107, miR-10a, miR-10b, miR-122, miR-1224-5p, miR-1226, miR-1227, miR-1228, miR-124, miR-1246, miR-1249, miR-1254, miR-125a-5p, miR-125b, miR-126, miR-1268, miR-127-3p, miR-1274b, miR-128, miR-1284, miR-1290, miR-1291, miR-129-3p, miR-1294, miR-1301, miR-1303, miR-1304, miR-1306, miR-1307, miR-1308, miR-130a, miR-130b, miR-133a, miR-133b, miR-134, miR-139-3p, miR-139-5p, miR-140-3p, miR-140-5p, miR-142-3p, miR-142-5p, miR-143, miR-145, miR-146a, miR-146b-3p, miR-146b-5p, miR-148a, miR-148b, miR-150, miR-151-3p, miR-151-5p, miR-152, miR-15a, miR-15b, miR-16, miR-17, miR-181a, miR-181b, miR-181d, miR-182, miR-185, miR-186, miR-1908, miR-191, miR-192, miR-193a-5p, miR-193b, miR-194, miR-197, miR-199a-3p, miR-199b-3p, miR-19b, miR-204, miR-206, miR-21, miR-210, miR-214, miR-22, miR-221, miR-222, miR-223, miR-23a, miR-23b, miR-24, miR-25, miR-26a, miR-26b, miR-27a, miR-28-3p, miR-296-5p, miR-29a, miR-29b, miR-29c, miR-30a, miR-30b, miR-30d, miR-30e, miR-31, miR-32, miR-320a, miR-320b, miR-320c, miR-320d, miR-323-3p, miR-323-5p, miR-324-3p, miR-324-5p, miR-326, miR-328, miR-329, miR-330-3p, miR-331-3p, miR-339-3p, miR-339-5p, miR-33a, miR-340, miR-342-3p, miR-342-5p, miR-34c-5p, miR-361-3p, miR-361-5p, miR-365, miR-375, miR-378, miR-382, miR-409-3p, miR-423-3p, miR-423-5p, miR-424, miR-425, miR-432, miR-433, miR-451, miR-483-3p, miR-483-5p, miR-484, miR-485-3p, miR-485-5p, miR-486-3p, miR-486-5p, miR-490-3p, miR-494, miR-499-5p, miR-532-3p, miR-543, miR-574-3p, miR-584, miR-589, miR-625, miR-629, miR-652, miR-671-3p, miR-7, miR-708, miR-720, miR-744, miR-760, miR-766, miR-874, miR-885-3p, miR-885-5p, miR-886-5p, miR-9, miR-923, miR-92a, miR-92b, miR-93, miR-935, miR-940, miR-941, miR-942, miR-98, miR-99a.
According to above-mentioned Solexa result, select the miRNAs satisfy following condition further to verify with the qRT-PCR method: 1) two groups multiple difference reach 5 times miRNA as the present invention in the preliminary serum biomarker of examination; 2) these miRNAs at least the copy number in one group of research object (" cases of cervical cancer " group or " healthy women contrast " group) greater than 50 with the raising detection efficiency.
The miRNAs that satisfies above-mentioned condition comprises: miR-1, miR-21, miR-23b, miR-28-3p, miR-29a, miR-100, miR-122, miR-125b, miR-181a, miR-206, miR-483-5p.QRT-PCR found that in 60 routine samples have the expression of 2 kinds of miRNAs (miR-21, miR-29a) in " cases of cervical cancer " group and " healthy women contrast " group to have significant difference.
Logistic Regression Analysis result shows that the expression level of these 2 kinds of miRNAs all exists remarkable related with the morbidity of cervical cancer: 2 kinds of miRNAs are high expression level in case all.
According to the above results, these 2 kinds of miRNAss relevant with cervical cancer pathogenesis are further detected in 45 routine precancerous lesions of uterine cervix cases (15 routine CIN I phase cases, 15 routine CIN II phase cases, 15 routine CIN III phase cases).We find that the expression of miR-21 and miR-29a is associated with the progress of CIN, specifically, miR-21 and the miR-29a expression in CIN I/II/III phase case serum/plasma all presents the rule of CIN I＜CIN II＜CIN III phase, and wherein the difference of miR-21 in several groups is greater than miR-29a.
The combination of further analyzing these 2 kinds of miRNA is used for the effect of cervical cancer and the auxiliary early diagnosis of precancerous lesion thereof, find that its combination is to the AUC[Area Under the ROC Curve of cervical cancer and precancerous lesion early diagnosis thereof, the lower area of ROC curve (Receiver Operating Characteristic Curve, experimenter's performance curve)] compare increase with miR-21 is single.
According to above-mentioned experimental result, the inventor has prepared the test kit that a kind of energy is used for the monitoring of precancerous lesions of uterine cervix progress, comprises to measure stable existence in experimenter's serum/plasma and detectable ripe miR-21 separately and primer and other detection reagent that miR-21 and miR-29a make up.
Particularly, miR-21 separately and the combination of miR-21 and miR-29a, perhaps miR-21 separately and the dependent diagnostic test kit of the primer formation that makes up of miR-21 and miR-29a help precancerous lesions of uterine cervix patient's dynamic monitoring, quick and precisely grasp patient's morbid state and coincident with severity degree of condition for the clinician, in time take the scheme of preventing and treating of more personalized to provide support, thereby avoid to greatest extent over-treatment, and the generation of prevention cervical cancer.
Serum/plasma refers to serum and plasma in this specification sheets, perhaps refers to serum or blood plasma.
Beneficial effect of the present invention:
Serum/plasma miRNA provided by the invention (microRNAs/miRNAs) mark is as the superiority of the mark of cervical cancer and the auxiliary early diagnosis of precancerous lesion thereof:
(1) serum/plasma miRNA serum s is a kind of new bio mark, be different from the traditional biological mark, not only stable, Wicresoft, be easy to detect, and quantitatively accurately, susceptibility and the specificity of medical diagnosis on disease will be improved greatly, the develop of such microRNA biomarker helps the auxiliary early diagnosis of cervical cancer and precancerous lesion thereof, for the development of other diseases biomarker is offered reference.
(2) serum/plasma miRNA serum s test kit is a kind of system, comprehensive auxiliary diagnosis and monitoring reagent box, can be used for the dynamic monitoring of the diagnosis of cervical cancer patient and precancerous lesion patient's progression of disease, help to reflect the morbid state of cervical cancer patient, understand precancerous lesions of uterine cervix patient disease progress, for the clinician quick and precisely grasps conditions of patients, in time takes the scheme of preventing and treating of more personalized to provide support.
(3) adopt tight design and appraisement system, inventor's initial stage adopts Solexa genome sequencing technology that serum/plasma miRNA serum s is carried out direct Sequencing obtaining the serum/plasma miRNA serum s express spectra of the special and unconventionality expression of disease, and the method for using qRT-PCR is verified in CINI/II/III phase case; The application acceleration of above method and strategy and the application that has guaranteed serum/plasma miRNA serum s biomarker and diagnostic kit also are the development supplying method of other diseases biomarker and the reference on the strategy.
The present invention is by the influence factor of control age etc. to disease progression, the research serum/plasma miRNA serum is in the application prospect of auxiliary cervical cancer and the auxiliary early diagnosis of precancerous lesion thereof, set forth the miRNAs of unconventionality expression for the impact of cervical cancer and precancerous lesion progress, disclose its examination and diagnostic value.Therefore, the present invention has obtained the relevant serum/plasma miRNA serum s expression database of cervical cancer pathogenesis and mark; Development and application by serum/plasma miRNA serum s biomarker and diagnostic kit, can be so that the auxiliary early diagnosis of cervical cancer and precancerous lesion thereof, and the precancerous lesions of uterine cervix dynamic monitoring is more convenient and easy, for the clinician quick and precisely grasps conditions of patients, for the clinical therapeutic efficacy evaluation lays the foundation, and for finding that the new small molecule drug provision with potential therapeutic value helps.
Description of drawings
Fig. 1: show miR-21, the miR-29a expression level in healthy women control group, cases of cervical cancer group, CIN I phase, CINII phase, CIN III phase case group.
The expression that shows miRNA distributes, and transverse axis represents research object, and the longitudinal axis represents the expression level of miRNA (with 2
-Δ CtExpression).The cases of cervical cancer group is compared with the healthy women control group, the expression level of miR-21, the miR-29a (miR-21:P=2.07994E-09 that all significantly raises; MiR-29a:P=0.000205).The expression of miRNA has the trend that progressively increases in the CIN I/II/III phase case group.
Fig. 2: show the independent ROC curve of miR-21.
Show that cases of cervical cancer group, CIN I phase, CIN II phase, CIN III phase case group are respectively the ROC curve of reference to the healthy women control group.Wherein:
---distinguish cases of cervical cancer group and control group: AUC 90.00%, sensitivity: 83.33%, specific degree: 96.67%;
----distinguish CIN III phase case group and control group: AUC 88.33%, sensitivity: 80.00%, specific degree: 96.67%;
--distinguish CIN II phase case group and control group: AUC 61.67%, sensitivity: 26.67%, specific degree: 96.67%;
... ... .. distinguishes CIN I phase case group and control group: AUC 51.67%, sensitivity: 6.67%, and specific degree: 96.67%.
Fig. 3: the ROC curve that shows miR-21 and miR-29a combination.
Show that cases of cervical cancer group, CIN I phase, CIN II phase, CIN III phase case group are respectively the ROC curve of reference to the healthy women control group.Wherein:
---distinguish cases of cervical cancer group and control group: AUC 95.83%, sensitivity: 93.33%, specific degree: 96.67%;
----distinguish CIN III phase case group and control group: AUC 89.56%, sensitivity: 80.00%, specific degree: 96.67%;
--distinguish CIN II phase case group and control group: AUC 68.56%, sensitivity: 40.00%, specific degree: 96.67%;
... ... .. distinguishes CIN I phase case group and control group: AUC 51.67%, sensitivity: 6.67%, and specific degree: 96.67%.
Embodiment
The invention will be further elaborated by the following examples.
The collection of embodiment 1 sample and the arrangement of sample data
The contriver began to have collected a large amount of cervical cancer patients and the peripheral blood sample of contrast from No.1 Attached Hospital, Nanjing Medical Univ, Nantong tumour hospital and Jiangning, Nanjing community so far 2006 month March, by the arrangement to the sample data, the contriver has therefrom selected 105 examples to meet the laboratory sample that sample checks order as Solexa and follow-up a series of qRT-PCR verifies of following standard:
1, new cases of cervical cancer;
2, perform the operation and chemicotherapy without crossing before the blood sampling, without chemicotherapy before the operation;
3, the healthy women with the case age-matched contrasts;
4, the CIN I/II/III phase case of age-matched.
And system acquisition the situations such as the demography data of these samples, clinical data.
The Solexa of miRNA order-checking experiment in embodiment 2 serum/plasma
In above-mentioned qualified 30 routine cervical cancer patients and 30 routine healthy women contrasts, two groups of age-matched.These two groups of crowds are obtained correlated results through Solexa order-checking test.Concrete steps are:
1, gets respectively " cases of cervical cancer " group and " healthy women contrast " group patient's serum 50ml, add isopyknic Trizol reagent;
2, be separated: room temperature is placed 15min, and the volume ratio of then pressing 0.2ml chloroform/1ml Trizol reagent adds chloroform, concussion 15s, room temperature 15min, 12,000g, 4 ℃, centrifugal 15min;
3, water is transferred to the centrifuge tube of new 50ml, 3 step phenol/chloroform are except the Deproteinization phase;
4, RNA precipitation: water is transferred in the new centrifuge tube, pressed 0.5ml Virahol/1ml Trizol reagent volume and add Virahol, preserve 60min, 12,000g, 4 ℃, centrifugal 60min for-20 ℃;
5, with the resuspended precipitation of 1ml Trizol reagent, suspension is transferred in the centrifuge tube of new 1.5ml;
6, repeated for 2,4 steps (the centrifugal 15min that changes into of the 4th step);
7, RNA washing: remove supernatant, add 75% ethanol, 12,000g, 4 ℃ of centrifugal 5min;
8, measure concentration: usually can obtain～5 μ g RNA/50ml serum;
9, total RNA carries out PAGE electrophoresis recovery 17-27nt RNA molecule;
10, will link primer (adaptor prime, Solexa check order universal primer) enzyme be associated in 3 of small RNA molecular ' with 5 ' end;
11, carry out after the RT-PCR reaction and check order;
12, data analysis and processing: in " cases of cervical cancer " group and " healthy women contrast " group, use the miRNA of the serum differential expression of Solexa sequence measurement discovery to enumerate out hereinbefore.
The qRT-PCR of miRNA experiment in embodiment 3 serum/plasma
According to above-mentioned Solexa result, the miRNA that following condition is satisfied in selection further verifies with the qRT-PCR method: 1) two groups of medium multiple differences reach 5 times miRNA as the present invention in the serum biomarker of preliminary examination, 2) among these miRNA at least the copy number in a group (" cases of cervical cancer " group and " healthy women contrast " group) greater than 50 with the raising detection efficiency.The miRNAs that satisfies above-mentioned condition comprises: miR-1, miR-21, miR-23b, miR-28-3p, miR-29a, miR-100, miR-122, miR-125b, miR-181a, miR-206, miR-483-5p.Primer (seeing Table 4) according to selected miRNAs design reverse transcription and qRT-PCR.The single individuality of serum of " cases of cervical cancer " group and " healthy women contrast " group is carried out the qRT-PCR detection of miRNA.In whole research process, all implement strict Quality Control.Each sample continuous detecting three times.All detect and all to adopt blind method, namely finish to avoid bias in the situation of sample background not knowing.
(1) preparation cDNA sample: a) get 500 μ l serum; B) add isopyknic water-saturated phenol, the vibration mixing, 4 ℃, centrifugal 3 minutes of 13200rpm gets supernatant; C) supernatant+1/2 volume (250 μ l) phenol+1/2 volume (250 μ l) chloroform, the vibration mixing, 4 ℃, centrifugal 3 minutes of 13200rpm gets supernatant; D) add and the isopyknic chloroform concussion of supernatant mixing, 4 ℃, centrifugal 3 minutes of 13200rpm gets supernatant as the RNA sample; E) then obtain cDNA by the RNA reverse transcription reaction.The reaction system of reverse transcription comprises one or more mixture of 4 μ l, 5 * AMV damping fluid, 2 μ l 10mM dNTP mixtures (Takara company), 0.5 μ l RNase inhibitor (Takara company), 2 μ l AMV (Takara company) and 1.5 μ l miRNA specific reverse primers.Reactions steps is 16 ℃ hatched 15 minutes, and 42 ℃ were reacted 1 hour, and hatched 5 minutes for 85 ℃;
(2) q-PCR: cDNA is pressed 1/5 volume dilution, get the cDNA after 1 μ l dilutes, add 0.3 μ l Taq enzyme (Takara company), 1 μ l, 20 * EVA GREEN, 0.2 μ l 10 μ M forward primers are a kind of, 0.2 the general reverse primer of μ l 10 μ M (URP:TGGTGTCGTGGAGTCG, SEQ ID No.23), 1.2 μ l 25mM MgCl
2, 1.6 μ l 2.5mM dNTP mixtures (Takara company), 2 μ l, 10 * PCR damping fluid, 13.5 μ lH
2O, 20 μ l systems are carried out q-PCR.What instrument used is ABI Prism 7300 quantitative real time PCR Instruments, and the reaction conditions of PCR is: carried out 1 circulation → 95 ℃, 15 seconds in 95 ℃, 5 minutes, carried out 40 circulations in 60 ℃, 1 minute.The expression amount ratio of two groups of sample serum miRNAs can be used equation 2
-Δ CtExpression, wherein Δ Ct=C
The T sample-C
The T confidential reference items, we detect at each, and adding six parallel shared Healthy Peoples contrast sample as reference in the version, calculate relative expression quantity.
QRT-PCR found that in 60 routine samples have the expression of 2 kinds of miRNAs (miR-21, miR-29a) between two groups to have significant difference (table 1).
Table 1.miRNA-21 and miRNA-29a qRT-PCR result
MiRNA further research of qRT-PCR experiment in the CIN case in embodiment 4 serum/plasma
According to the above results, these 2 kinds of miRNAss relevant with cervical cancer pathogenesis are further detected in CIN I/II/III phase patient.We find miR-21, the expression of miR-29a in CIN case group serum all is significantly higher than control group, and mR-21 expresses with miR-29a and presents consistent trend with the progress of CIN among the patients serum of precancerous lesions of uterine cervix, and affiliated CIN is by stages higher, its expression higher (table 1 and Fig. 1).
Embodiment 5miR-21 separately and the combination of miR-21 and miR-29a to diagnosis and the ROC curve thereof of cervical cancer pathogenesis
According to above-mentioned Real-time PCR result, the inventor passes through 5 groups of plasma samples (" cases of cervical cancer group ", " healthy women control group ", " CIN I phase case group ", " CIN II phase case group ", " CIN III phase case group ") the analysis of miRNAs expression level, respectively take 95 percentiles of healthy women control group miR-21 and miR-29a expression amount as threshold value, to miR-21, miR-29a marks, expression amount is 0 minute less than the scoring of 95 percentiles, expression amount is 1 minute more than or equal to the scoring of 95 percentiles, further try to achieve PTS and (see Table 2, table 3), draw susceptibility and the specificity that the ROC curve is assessed prediction with this, and then assess the judgement that miR-21 combination high expression level independent and miR-21 and miR-29a is fallen ill in early days to cervical cancer.The ROC analytical results shows, miR-21 opens healthy women control group and cases of cervical cancer component with 90.0% AUC (ROC area under curve) separately, and the sensitivity of best stagnation point is 83.33%, and specific degree is 96.67%; AUC with 88.33% opens healthy women control group and CIN III phase case component, and the sensitivity of best stagnation point is 80.00%, and specific degree is 96.67%; AUC with 61.67% distinguishes healthy women control group and CIN II phase case group, and sensitivity is 26.67%, and specific degree is 96.67%; AUC with 51.67% distinguishes healthy women control group and CIN I phase case group, and sensitivity is 6.67%, and specific degree is 96.67% (Fig. 2).The combination of miR-21 and miR-29a is opened healthy women control group and cases of cervical cancer component with 95.8% AUC (ROC area under curve), and the sensitivity of best stagnation point is 93.33%, and specific degree is 96.67%; AUC with 89.6% opens healthy women control group and CIN III phase case component, and the sensitivity of best stagnation point is 80.00%, and specific degree is 96.67%; AUC with 68.6% distinguishes healthy women control group and CIN II phase case group, and sensitivity is 40.00%, and specific degree is 96.67%; AUC with 51.7% distinguishes healthy women control group and CIN I phase case group, and sensitivity is 6.67%, and specific degree is 96.67% (Fig. 3).
Therefore, the inventor has proved and has adopted miR-21 combination independent and miR-21 and miR-29a well healthy women contrast and cervical cancer patient to be distinguished, and the severity of this differentiation and CIN presents good consistence.
Table 2.miRNA-21 expression level score
The score summation of table 3.miRNA-21 and miRNA-29a
The making of miRNA test kit and operating process are based on solexa order-checking, the technology such as RT-PCR and real-time PCR.
Test kit comprises that (primer of miR-21 is SEQ ID No.3 and SEQ IDNo.4 to the serum/plasma miRNA serum primer; The primer of miR-29a is SEQ ID No.11 and SEQ ID No.12), required enzyme commonly used and/or the reagent of corresponding PCR reaction can also be arranged, as: reversed transcriptive enzyme, damping fluid, dNTPs, MgCl2, DEPC water, fluorescence dye or probe, the Taq enzyme, general reverse primer (URP:TGGTGTCGTGGAGTCG, SEQ ID No.23) etc., can select according to the experimental technique of concrete employing, these enzymes commonly used and/or reagent are well known to those skilled in the art, standard substance and contrast (such as normal people's sample of quantitative mark etc.) and normal reference value (miR-21:5.0948, miR-29a:3.8238) can also be arranged in addition.The value of this test kit is only to need serum/plasma and does not need other tissue sample, detect the variation tendency of miRNA by the fluorescence of simplifying most or probe method, again by the auxiliary early diagnosis cervical cancer of this trend and precancerous lesion thereof, not only stable, easy to detect, and quantitatively accurately, greatly improve susceptibility and the specificity of medical diagnosis on disease, therefore this test kit is dropped into practice, can help to instruct the clinical auxiliary early diagnosis of accurately making.
Claims (4)
1. the application of human serum/blood plasma miRNA mark in preparation human hela and the auxiliary early diagnosis kit of precancerous lesion thereof, this mark is the miR-21 in human serum/blood plasma source and the combination of miR-29a.
2. the application of the primer of a human serum/blood plasma miRNA mark in preparation human hela and the auxiliary early diagnosis kit of precancerous lesion thereof, this primer is: the primer sequence of miR-21 is shown in SEQ ID No.3 and SEQ ID No.4; The primer sequence of miR-29a is shown in SEQ ID No.11 and SEQ ID No.12.
3. the auxiliary early diagnosis kit of a human hela and precancerous lesion thereof, it is characterized in that this test kit contains the primer of miR-21 and miR-29a combination among the human serum/blood plasma miRNA, wherein: the primer sequence of miR-21 is shown in SEQID No.3 and SEQ ID No.4; The primer sequence of miR-29a is shown in SEQ ID No.11 and SEQ ID No.12.
4. described diagnostic kit according to claim 3 is characterized in that this test kit also comprises PCR reaction enzyme and/or reagent commonly used.
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Cited By (1)
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101622349A (en) * | 2006-12-08 | 2010-01-06 | 奥斯瑞根公司 | miR-20 regulated genes and pathways as targets for therapeutic intervention |
| CN101709328A (en) * | 2009-12-10 | 2010-05-19 | 浙江理工大学 | Serology biological marker for detecting tumor of breast and application thereof |
| CN101755208A (en) * | 2007-07-25 | 2010-06-23 | 路易斯维尔大学研究基金会公司 | Allochthon associated microRNA as diagnostic marker |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2471924A1 (en) * | 2004-05-28 | 2012-07-04 | Asuragen, INC. | Methods and compositions involving microRNA |
| US20080076674A1 (en) * | 2006-07-06 | 2008-03-27 | Thomas Litman | Novel oligonucleotide compositions and probe sequences useful for detection and analysis of non coding RNAs associated with cancer |
| US8361714B2 (en) * | 2007-09-14 | 2013-01-29 | Asuragen, Inc. | Micrornas differentially expressed in cervical cancer and uses thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101622349A (en) * | 2006-12-08 | 2010-01-06 | 奥斯瑞根公司 | miR-20 regulated genes and pathways as targets for therapeutic intervention |
| CN101755208A (en) * | 2007-07-25 | 2010-06-23 | 路易斯维尔大学研究基金会公司 | Allochthon associated microRNA as diagnostic marker |
| CN101709328A (en) * | 2009-12-10 | 2010-05-19 | 浙江理工大学 | Serology biological marker for detecting tumor of breast and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019022605A1 (en) * | 2017-07-25 | 2019-01-31 | Stichting Vumc | Micro rna-based methods and compositions for detection of hpv-induced invasive cancers, and their high-grade precursor lesions |
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