CN102933719B - Compositions and methods for microrna expession profiling in plasma of colorectal cancer - Google Patents

Abstract

The present invention relates to compositions and methods for microRNA (miRNA) expression profiling in plasma of colorectal cancer. In particular, the invention relates to a diagnostic kit of molecular markers in blood for diagnosing colorectal cancer, monitoring the cancer therapy and/or treating colorectal cancer, the kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in plasma of colorectal cancer and healthy control plasma, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative for the presence of colorectal cancer. The invention further relates to corresponding methods using such nucleic acid expression signatures for identifying colorectal cancer as well as for preventing or treating such a condition. Finally, the invention is directed to a pharmaceutical composition for the prevention and/or treatment of colorectal cancer.

Description

Compositions and methods for microRNA expression profiling in colorectal cancer plasma

field of invention

The present invention relates to compositions and methods for microRNA (microRNA) expression profile analysis in colorectal cancer (colorectal cancer) plasma.

Background of the invention

Colorectal cancer (CRC) is the most important human cancer with ~1,041,200 new cases worldwide in 2008. It is the third most common cancer and the fourth leading cause of cancer death in the world. (Gry. R. et al. (1997)) Curr Probl Cancer 21, 233-300; Petersen, G. M. et al. (1999) Cancer 86, 254002550). CRC is curable if diagnosed in the early stages. In the early stage, most patients have no clinical symptoms of the disease, so early screening for CRC is necessary. Evidence shows that early detection of colorectal cancer can greatly reduce the incidence and mortality of the disease (Anwar, R. (2006) Digestive and Liver Disease 34, 279-282).

Initially CRC is characterized by hyperproliferation (atypia) of the colonic epithelium, which transforms first into an inflammatory adenomatous polyp and then into an abnormal neoplastic adenoma (ie, a benign tumor) of the colorectal mucosa. Typically, only a small fraction of formed adenomas (60-70% in patients aged 60 years) develop into malignant adenocarcinoma. More than 95% of CRC cases present as adenocarcinoma (Muto, T. et al. (1975) Cancer 36, 2251-2270; Fearon, E.R. and Vogelstein, B. (1990) Cell 61, 759-767).

Existing standard screening methods for CRC include colonoscopy and fecal occult blood testing. But both tests have serious flaws. Colonoscopy is effective, but most people are reluctant to undergo the test because of its high cost, great discomfort, and potential for more side effects. Fecal occult blood testing, on the other hand, is simple and inexpensive but relatively inaccurate. However, there are currently no specific molecular markers that allow reliable diagnosis of CRC, especially for CRC presenting as adenocarcinoma and/or for benign adenoma developing into malignancy.

Therefore, the discovery of new biomarkers has the most important clinical utility, especially these markers for early diagnosis of tumor development in order to allow early treatment of cancer and avoid unnecessary surgical intervention. Ideally, these markers should enable the detection of cancer at a stage where malignant cells are not detectable by in situ techniques or microscopic analysis of biopsies or resections.

Some diagnostic methods are also limited because their analysis is based on a single molecular marker, which affects the reliability and/or precision of the test. Furthermore, a single marker often cannot predict in detail the relevant latency stage, tumor progression and similar. Therefore, the discovery of alternative molecular markers and detection modalities to overcome these obstacles awaits.

A solution to this problem may be based on small regulatory RNA molecules, especially microRNAs (miRNAs), which constitute evolutionarily conserved endogenously expressed small untranslated 20-25 nucleotide (nt) in length RNA. Since its discovery 10 years ago, it is believed that it can mediate the expression of target mRNA and thus have important functions involved in cell generation, differentiation, proliferation and apoptosis. (Bartel, D.P. (2004) Cell 116, 281-297, Ambros, V. (2004) Nature 431, 350-355; He. L. et al. (2004) Nat Rev Genet 5, 522-531). In addition, as 25 cancer biomarkers, miRNA has advantages over mRNA due to its stability in vitro and long life in vivo (Lu, J. et al. (2005) Nature 435, 834-838; Lim, L.P. et al. (2005 ) Nature 433, 769-773).

miRNAs originate from stem-loop precursors (pre-miRNAs) processed by RNase III Drosha during primary transcription. After being transported to the cytoplasm, another RNaseIII called Dicer cuts the pre-miRNA hairpin into short double-stranded RNA (dsRNA), one of which becomes the mature miRNA in the miRNA protein (miRNP). miRNAs direct miRNPs to their functional target mRNAs (Bartel, D.P. (2004) Cell 23, 281-292; He, L. and Hannon, G.J. (2004) Nat Rev Genet 5, 522-531).

Depending on the degree of complementarity between the miRNA and the target, miRNAs can direct different regulatory processes. Target mRNAs that are highly complementary to miRNAs are specifically cleaved by the same structure as interfering RNA (RNAi). Thus, in this context, miRNAs function as small interfering RNAs (siRNAs). Target mRNAs that are less complementary to miRNAs are also directed to cellular degradation pathways or are translationally repressed without affecting mRNA levels. However, the mechanism by which miRNAs inhibit the translation of target mRNAs remains controversial. Existing data suggest that miRNAs may function as oncogenes or tumor suppressor genes in cancer. For example, overexpressed mir-17-92 in cancer may function as an oncogene and may function as a tumor suppressor gene and/or control cell differentiation or Apoptotic genes thus promote cancer development. Similarly, low expression of let-7a, which functions as a tumor suppressor gene, may inhibit cancer by regulating oncogenes and/or genes controlling cell differentiation or apoptosis (Zhang, B. (2007) Dev Biol 302, 1-12 ). suggest that they play a role in cancer initiation and progression.

High-throughput miRNA quantification technologies such as miRNA microarrays, real-time RT-PCR-based TaqMan miRNA assays, etc. provide a powerful means to study the overall miRNA profile of the entire cancer genome. Accumulating evidence indicates that multiple miRNAs are abnormally expressed in human cancers including leukemia, lymphoma, glioblastoma, colon, lung, breast, prostate, thyroid, liver, and ovarian cancers, and in normal The expression in tissues and cancer tissues is different. (Zhang, L. 25 (2008) Adv Exp Med Biol 622, 69-78). Therefore, miRNA profiling is used as a marker for the discovery of various cancers, suggesting that it will help to further define molecular diagnosis, prognosis and treatment. Aberrant miRNA expression in human cancers suggests the potential of these miRNAs as biomarkers and molecular therapeutic targets.

Among the many possible types of samples, blood is considered an ideal sample for screening high-risk individuals, as it can be collected in a minimally invasive manner, allowing early detection, diagnosis, monitoring and effective treatment of cancer. It has been shown that tumor-derived miRNA can exist in human plasma or serum in a very stable form under the protection of endogenous RNase. These tumor-derived miRNAs in plasma or serum can be used as biomarkers for discovering cancer at measurable levels. In addition, the correlation between plasma and serum miRNA levels is large, indicating that plasma or serum samples can be used as a sample selection type for clinical use of miRNA as a biomarker for cancer prognosis (Mitchell, P.S. et al. (2008) Proc Natl Acad Sci USA 105, 10513-10518; Gilad, S. et al. (2008) PLoSONE 3, e3148; Chen, X. et al. (2008) Cell Res 18, 997-101006).

Several studies have reported miRNA expression profiles in plasma or serum of human colorectal cancer cases (Chen, X. (2008) Cell Res 18, 997-1006; Ng, E.K.O. (2009) Gut 58, 1375-1381; Huang, Z.2009 , Int J Cancer, published on line). More than 100 circulating miRNAs were found in healthy individuals (Mitchell, P.S. (2008) Proc Natl Acad Sci USA105, 10513-10518), and this profile is very similar to that of colorectal cancer patients with multiple tumor-specific miRNAs different. Chen et al. proved that 69 miRNAs existed in the serum of colorectal cancer patients but not in the normal control group (Chen, X. (2008) Cell Res 18, 997-1006). Furthermore, they found a unique expression profile of 14 miRNAs present in the serum of colorectal cancer but not in another cancer group (lung cancer). The study by Ng et al. showed that miR-92 was significantly elevated in the plasma of CRC cases and has potential as a non-invasive molecular marker for CRC screening (E.K.O. (2009) Gut 58, 1375-1381). Furthermore, Huang et al. found that plasma miR-29a has great potential as a new non-invasive biomarker for early detection of CRC (Huang, Z. 2009, Int J Cancer. published on line). However, another study found that has-miR-92a was significantly decreased in the plasma of patients with acute leukemia, and the ratio of miR-92a/miR-638 in plasma has great potential as a new biomarker for detecting leukemia (Tanaka, M.et al. (2009) PLoS ONE 4, e5532). However, due to the lack of sufficient sensitivity and specificity, a single miRNA is not suitable for clinical application as an accurate diagnostic biomarker.

Therefore, it is still necessary to discover a panel of diagnostic miRNA markers in the plasma or serum of colorectal cancer patients. The determination of blood-based miRNA profiles (signatures) incorporating multiple miRNA biomarkers will enable a non-invasive, rapid, accurate and economical method of identifying colorectal cancer patients. In addition, there is a continuing need for corresponding approaches to early stage ie colorectal cancer screening, early detection of cancer recurrence, and/or monitoring of cancer therapy in high risk individuals.

Invention purpose and overview

The object of the present invention is to provide a new method for diagnosing colorectal cancer, monitoring cancer treatment, and/or treating colorectal cancer, wherein by measuring multiple nucleic acid molecules in blood, each nucleic acid molecule encoding microRNA (miRNA) sequence, wherein one or more of said plurality of nucleic acid molecules is differentially expressed in plasma of colorectal cancer compared to healthy controls and/or compared to healthy individuals, hepatocellular carcinoma and lung cancer, wherein said one or A plurality of differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative of the presence of colorectal cancer, wherein the nucleic acid expression signature includes a tumor-related signature and a plasma-related signature. specific signature).

Furthermore, it is an object of the present invention to provide a corresponding method for identifying one or more nucleic acid expression signatures in blood exhibiting colorectal cancer. More specifically, it is an object of the present invention to provide a method for differentiating colorectal cancer compared to healthy controls, and/or healthy individuals, hepatocellular carcinoma and lung cancer.

These and other objects will become apparent from the following description, which are achieved by the subject-matter of the independent claims. Some preferred embodiments of the invention are then defined by the subject-matter of the dependent claims.

In a first aspect, the present invention relates to a diagnostic kit for identifying one or more molecular markers in blood of target plasma exhibiting colorectal cancer, said kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding RNA sequences, wherein one or more of said plurality of nucleic acid molecules are differentially expressed in target plasma and in one or more control plasmas, wherein said one or more differentially expressed nucleic acid molecules are derived from tumor-associated or plasma-specific wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative of the presence of colorectal cancer.

The nucleic acid expression signature defined herein may comprise at least 35 nucleic acid molecules, preferably at least 12 nucleic acid molecules, particularly preferably at least 6 nucleic acid molecules.

In a preferred embodiment, the nucleic acid expression characteristics comprise at least one nucleic acid molecule encoding a microRNA sequence whose expression is up-regulated in one or more target plasma compared with one or more healthy controls; and comprising at least one A nucleic acid molecule encoding a microRNA sequence whose expression is down-regulated in one or more target plasma compared to one or more healthy controls.

In a preferred embodiment, the nucleic acid expression signature comprises encoding tumor-associated signatures hsa-miR-409-3p, hsa-miR-25, hsa-miR-93, hsa-miR-96, hsamiR-301a, hsa-miR -342-3p, hsa-miR-19b, hsa-miR-451, hsa-miR-486-5p, hsamiR-187*, hsa-miR-92a, hsa-miR-19a, hsa-miR-20b, hsa- miR-20a, hsa-miR-139-3p, hsa-miR-107, hsa-miR-17, hsa-miR-140-3p, hsa-miR-30e, hsa-miR-185; and plasma-specific signature hsa -mir-671-3p, hsa-mir-16-2*, hsa-miR-30c-1*, hsa-miR-548c-5p, hsa-miR-16, hsa-miR-15a, hsa-miR-425 , hsa-let-7i, hsamiR-363, hsa-miR-15b, hsa-miR-101, hsa-miR-190b, hsa-miR-130a, hsa-miR-331-3p, hsa-miR-345; and Any one or more nucleic acid molecules of internal stability control hsa-miR-1238 and hsa-miR-1228.

Particularly preferably, compared with one or more healthy controls, in the one or more target plasma: any one or more nucleic acids encoding hsa-miR-409-3p and hsa-mir-671-3p Molecule expression was upregulated; while encoding hsa-miR-25, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR- 451, hsa-miR-486-5p, hsa-miR-187*, hsa-miR-92a, hsa-miR-19a, hsa-miR-20b, hsa-miR-20a, hsa-miR-139-3p, hsa -miR-107, hsamiR-17, hsa-miR-140-3p, hsa-miR-30e, hsa-miR-185'hsa-mir-16-2*, hsa-miR-30c-1*, hsa-miR -548c-5p, hsa-miR-16, hsa-miR-15a, hsa-miR-425, hsa-let-7i, hsa-miR-363, hsa-miR-15b, hsa-miR-101, hsa-miR - The expression of any one or more nucleic acid molecules of 190b, hsa-miR-130a, has-miR-331-3p and hsa-miR-345 is down-regulated; hsa-miR-1238 and hsa-miR-1228 have no change.

In a more preferred embodiment, the nucleic acid expression signature comprises encoding tumor-associated signatures hsamiR-409-3p, hsa-miR-25, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR- 342-3p, hsa-miR-19b, hsa-miR-451 and encoding plasma-specific features hsa-mir-671-3p, hsa-mir-16-2*, hsa-miR-30c-1*, hsa-miR - any one or more nucleic acid molecules of 548c-5p.

Particularly preferably, compared with one or more healthy controls, in one or more target plasma: expression of any one or more nucleic acid molecules encoding hsa-miR-409-3p and hsa-mir-671-3p Upregulated; while encoding hsa-miR-25, hsamiR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsamir-16 The expression of any one or more nucleic acid molecules of -2*, hsa-miR-30c-1* and hsa-miR-548c-5p is down-regulated.

In another preferred embodiment, the nucleic acid expression signature comprises hsa-miR-409-3p, hsa-miR-25, hsa-miR-93, hsa-miR-96 and plasma-specific signature hsa-mir - Any one or more nucleic acid molecules of 671-3p, hsa-mir-16-2*.

Particularly preferably, compared with one or more healthy controls, in one or more target plasma: any one or more nucleic acid molecules encoding hsa-miR-409-3p and hsa-mir-671-3p The expression of any one or more nucleic acid molecules encoding hsa-miR-25, hsamiR-93, hsa-miR-96 and and hsa-mir-16-2* is down-regulated.

In a particularly preferred embodiment, the nucleic acid expression signature comprises hsa-miR-409-3p/hsa-miR-16-2*, hsa-miR-409-3p/hsa-miR-96, hsa-miR-671- 3p/hsa-miR-548c-5p, hsa-miR-671-3p/hsa-miR-16-2*, hsa-miR-671-3p/hsa-miR-30c-1*, hsa-miR-671- 3p/hsa-miR-342-3p, hsa-miR-671-3p/hsa-miR-96, hsa-miR-671-3p/hsa-miR-301a, hsa-miR-671-3p/hsa-miR- 345, hsa-miR-409-3p/hsa-miR-345, hsa-miR-409/hsa-miR-331-3p, hsa-miR-671-3p/hsa-miR-331-3p, hsa-miR- Any one or more nucleic acid molecule combinations of 671-3p/hsa-miR-25, hsa-miR-409-3p/hsa-miR-93 and hsa-miR-671-3p/hsa-miR-93.

In a second aspect, the present invention relates to a diagnostic kit for differentiating colorectal cancer from healthy individuals, hepatocellular carcinoma and lung cancer. The kit comprises a plurality of nucleic acid molecules, each nucleic acid molecule encodes a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules are in target plasma and in one or more healthy individuals, hepatocellular carcinoma and lung cancer differentially expressed, wherein said one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative of the presence of colorectal cancer.

A nucleic acid expression signature as defined herein may comprise at least 23 nucleic acid molecules, preferably at least 18 nucleic acid molecules, and more preferably at least 6 nucleic acid molecules.

In a preferred embodiment, the nucleic acid expression signature comprises at least one nucleic acid molecule encoding a microRNA sequence, whose expression in one or more target plasma is compared with one or more healthy individuals, hepatocellular carcinoma and lung cancer upregulated; and comprising at least one nucleic acid molecule encoding a microRNA sequence whose expression is downregulated in one or more target plasma compared to one or more healthy individuals, hepatocellular carcinoma, and lung cancer.

In a more preferred embodiment, the nucleic acid expression signature comprises the codes for: tumor-associated signature hsa25miR-409-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-7, hsa-miR-196b, hsa - miR-93, hsa-miR-486-5p, hsa-miR-25, hsa-miR-92a, hsa-miR-19b; plasma-specific signatures hsa-miR-671-3p and hsa-miR-16-2 *, hsa-miR-92b, hsa-miR-129*, hsa-miR-563, hsamiR-602, hsa-miR-1227, hsa-miR-196a and internal stable controls: hsa-miR-1238 and hsa-miR - any one or more nucleic acid molecules of 1228.

Particularly preferably, one or more target plasmas encode hsa-miR-409-3p, hsa-mir-671-3p, hsa-miR-33b*, hsa-miR compared to healthy individuals, hepatocellular carcinoma and lung cancer -92b, hsa-miR-149, hsa-miR-129*, hsa-miR-563, hsa-miR-129-3p, hsa-miR-634, hsa-let-7b*, hsa-miR-602, and hsa -The expression of any one or more nucleic acid molecules of miR-1227 is up-regulated, while encoding hsa-miR-16-2*, hsa-miR-7, hsa-miR-196a, hsamiR-196b, hsa-miR-486- The expression of any one or more nucleic acid molecules of 5p, hsa-miR-93, hsa-miR-25, hsa-miR-92a and hsa-miR-19b was down-regulated; hsa-miR-1238 and hsa-miR-1228 were not Variety.

In a more preferred embodiment, the nucleic acid expression signature comprises the encoding tumor-associated signature has-10miR-409-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-7 and the plasma-specific signature hsa - any one or more nucleic acid molecules of miR-671-3p and hsa-miR-16-2*.

Particularly preferably, hsa-miR-409-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-33b*, hsa- The expression of any one or more nucleic acid molecules of miR-671-3p is up-regulated; while the expression of any one or more nucleic acid molecules encoding hsa-miR-16-2* and hsa-miR-7 is down-regulated.

In a particularly preferred embodiment, the nucleic acid expression characteristics include encoding hsa-miR-33b*/hsa20-miR-196a, hsa-miR-92b/hsa-miR-196a, hsa-miR-563/hsa-miR-196a, hsa -miR-1227/hsa-miR-196a, hsa-miR-129-3p/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-16-2*, hsa-miR-92b/ hsa-miR-16-2*, hsa-miR-129*/hsa-miR-196a, hsa-miR-1227/hsa-miR-16-2*, hsa-miR-129-3p/hsa-miR-196a , hsa-miR-602/hsa-miR-196a, hsa-miR-33b*/hsamiR-16-2*, hsa-miR-563/hsa-miR-16-2*, hsa-miR-129*/hsa -miR-7, hsa-miR-33b*/hsa25-miR-7, hsa-miR-563/hsa-miR-7, hsa-miR-602/hsa-miR-16-2*, hsa-miR-129 -3p/hsamiR-7, hsa-miR-92b/hsa-miR-7, hsa-miR-1227/hsa-miR-7, hsa-miR-33b*/hsa-miR-196b, hsa-miR-1227/ Any one or more nucleic acid combinations of hsa-miR-196b, hsa-miR-92b/hsa-miR-196b and hsa-miR-602/has-miR-7.

In a third aspect, the present invention relates to a method for identifying one or more plasmas exhibiting colorectal cancer, said method comprising: (a) determining the expression levels of a plurality of nucleic acid molecules in said one or more target plasmas , each nucleic acid molecule encodes a microRNA sequence; (b) determines the expression level of said plurality of nucleic acid molecules in one or more healthy control plasma; (c) by comparing the obtained in steps (a) and (b) Respective expression levels, one or more nucleic acid molecules differentially expressed in the target plasma and control plasma are identified from the plurality of nucleic acid molecules, wherein the differentially expressed one or more nucleic acid molecules together represent A defined signature that is indicative of the presence of colorectal cancer.

In a preferred embodiment of the present invention, the method includes (a) determining the expression level of the combination of nucleic acid molecules in one or more target plasma, each nucleic acid molecule is encoded microRNA sequence, and calculated with a specific formula; (b ) determining the expression level of the combination of nucleic acid molecules in one or more healthy control plasma, and calculating with a specific formula; and (c) comparing the respective expression levels obtained in steps (a) and (b) to determine Identifying differences in said combination in said one or more target plasmas, wherein the one or more differentially expressed combinations represent features indicative of the presence of colorectal cancer.

In a more preferred embodiment, the method is further used to differentiate colorectal cancer from healthy individuals, hepatocellular carcinoma and lung cancer.

In a fourth aspect, the present invention relates to a method for monitoring treatment of colorectal cancer, the method comprising: (a) identifying nucleic acid expression signatures in one or more target plasmas by using the methods defined herein; and (b) in Monitoring the expression of one or more nucleic acid molecules encoding microRNA sequences included in the nucleic acid expression signature in the blood in such a way that the expression of the nucleic acid molecules whose expression in plasma was up-regulated before treatment The expression of nucleic acid molecules whose expression in plasma was downregulated before treatment is downregulated after treatment is upregulated after treatment.

In a fifth aspect, the present invention relates to a method for preventing or treating colorectal cancer, said method comprising: (a) identifying nucleic acid expression characteristics in plasma using the methods defined herein; and (b) altering said nucleic acid in blood The expression profile comprises the expression of one or more nucleic acid molecules encoding microRNA sequences, said alteration being carried out in such a way that the expression of nucleic acid molecules whose expression is up-regulated in blood is down-regulated, whereas the expression of nucleic acid molecules whose expression is up-regulated in blood Expression of a downregulated nucleic acid molecule is upregulated.

In a sixth aspect, the present invention relates to a pharmaceutical composition for preventing and/or treating colorectal cancer in blood, said composition comprising one or more nucleic acid molecules, each nucleic acid molecule coding sequence, said sequence being as As defined herein, the microRNA sequence encoded by the nucleic acid molecule whose expression is upregulated in the blood plasma from a colorectal cancer patient is at least partially complementary, and/or the sequence corresponds to the microRNA sequence as defined herein in a blood plasma from a colorectal cancer patient. A microRNA sequence encoded by a nucleic acid molecule whose expression is downregulated in plasma.

Finally, in the seventh aspect, the present invention relates to the use of the pharmaceutical composition in the preparation of a medicament for preventing and/or treating hepatocellular carcinoma.

Other embodiments of the invention will become apparent from the following detailed description.

Brief description of the drawings

Figure 1 : shows a flow chart which systematically illustrates the basic method steps for determining the expression signature with reference to the method of the present invention for identifying one or more target plasmas exhibiting colorectal cancer.

Figure 2: Depicts the determination according to the invention of one or more target plasma in colorectal cancer comprising human miRNAs in a particularly preferred expression profile in the first aspect. Expression levels and accuracy (RUC) (ie, up- or down-regulation) of these miRNAs were also illustrated in colorectal cancer patients compared to healthy controls.

Figure 3: Depicts the respective expression levels and ROC curve analysis of two upregulated (hsa-miR-409-3p and has-miR-671-3p) miRNAs in colorectal cancer progression from Dukes' A, B, C and D cancer group selected. Data were obtained separately from microarrays and normalized to the internal stability control has-miR-1238. The data demonstrate that Dukes' A stage colorectal cancer can be detected by the miRNA signature discovered in the present invention.

Figure 4: illustrates the human miRNAs included in the particularly preferred expression signature according to the second aspect of the invention, in order to further differentiate colorectal cancer from healthy controls, hepatocellular carcinoma and lung cancer. It was also shown that the expression levels and accuracy (AUC) (ie, up- or down-regulation) of these miRNAs were expressed in colorectal cancer patients compared with healthy controls, hepatocellular carcinoma, and lung cancer. The first combination (hsa20-miR-33b*/hsa-miR-196a) showed an accuracy of 85% (AUC=0.860) as a diagnostic biomarker for distinguishing CRC from healthy individuals, hepatocellular carcinoma and lung cancer.

Figure 5: Example of ROC curve analysis depicting two tumor-associated miRNAs (hsa-miR-93 and hsa-miR-25) obtained by quantitative RT-PCR in colorectal cancer and healthy control plasma (0: healthy individuals; 1: colorectal cancer). The results demonstrate the high sensitivity and specificity of these miRNAs as diagnostic biomarkers.

Detailed description of the invention

The present invention is based on the unexpected discovery that colorectal cancer can be reliably identified with high accuracy and sensitivity by specific miRNA expression signatures in plasma, wherein said expression signatures as defined herein typically include upregulated and downregulated Human miRNAs. More particularly, the miRNA expression signature—by analyzing the overall miRNA expression pattern and/or individual miRNA expression levels in plasma—enables the detection of HCC in early disease states and the identification of healthy individuals, hepatocellular carcinoma, and lung carcinoma.

The invention exemplified below may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The present invention will be described with reference to particular embodiments and with reference to the accompanying drawings but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are to be considered as non-limiting.

When the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined as comprising at least a certain number of embodiments, this is also understood to reveal a group which preferably consists only of these embodiments.

When an indefinite or definite article is used when referring to a singular noun eg "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

The term "about" in the present invention refers to the accuracy range understood by those skilled in the art that can still guarantee the technical effect of the objective feature. The term usually means ±10%, preferably ±5%, of the indicated value.

In addition, the terms first, second, third, (a), (b), (c), etc. are used in the description and claims to distinguish similar elements, and are not necessarily described in sequence or in time. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the described embodiments of the invention are capable of operation in other sequences than described or illustrated herein.

Further definitions of terms are given below when terms are used.

The following terms or definitions are provided for the understanding of the present invention only. These definitions should not be considered to have less scope than understood by those skilled in the art.

An object of the present invention is to provide a new method for diagnosing colorectal cancer, monitoring cancer treatment, and/or treating colorectal cancer by measuring multiple nucleic acid molecules in blood, each nucleic acid molecule encoding a microRNA (miRNA) sequence, wherein one or more of said plurality of nucleic acid molecules are differentially expressed in colorectal cancer plasma versus healthy individual, hepatocellular carcinoma and lung cancer plasma, wherein said one or more differentially expressed nucleic acid molecules together represent nucleic acid expression A signature, the nucleic acid expression signature is an indication for the presence of colorectal cancer, wherein the nucleic acid expression signature includes a tumor-associated signature and a plasma-specific signature.

The term "colorectum" herein refers to the colon, rectum and/or appendix, ie the entire large intestine.

Herein the term "cancer" (also referred to as carcinoma) generally refers to any type of malignant neoplasm, i.e. any morphology and appearance of target cells that exhibit or have a propensity to develop cancerous features compared to unaffected (healthy) wild-type control cells. / or physiological changes (based on genetic re-programming). Examples of such changes may relate to cell size and shape (increase in size or size), cell proliferation (increase in cell number), cell differentiation (change in physiological state), apoptosis (programmed cell death), or cell survival. Thus, the term "colorectal cancer" refers to cancerous growths in the colon, rectum, and appendix.

The most common colorectal cancer (CRC) cell type is adenocarcinoma, accounting for approximately 95%. Other types of CRC include lymphoma and squamous cell carcinoma, among others.

Colorectal cancer can be classified according to the Dukes system (Dukes, C.E. (1932) J. Pathol. Bacteriol. 35, 323-325). The following stages are included: Dukes A - tumor confined to the bowel wall; Dukes B - tumor invades through the bowel wall; Dukes C - tumor involves lymph nodes; Dukes D - tumor has distant metastasis.

The term "plasma" herein refers to the yellow liquid component of blood in which the blood cells of whole blood are usually suspended. About 55% of the total blood volume. It is mostly water (90% by volume) and contains dissolved proteins, glucose, coagulation factors, mineral ions, hormones and carbon dioxide (plasma is the main medium for the transport of secreted products). Plasma is prepared by centrifuging fresh blood in a centrifuge until the blood cells settle to the bottom of the centrifuge tube. The plasma is then purified and transferred. The density of plasma is about 1025kg/m3, or 1.025kg/l. Current studies indicate that miRNAs are stable in plasma. The term "plasma sample" refers to plasma obtained from an individual being tested or a healthy control.

The term "patient" as used herein refers to a person who should at least be considered to have hepatocellular carcinoma; the term "target plasma" as used herein refers to plasma obtained from a patient; the term "healthy individual" or "healthy control" specifically refers to a person who is not Healthy individuals with any manifestation of cancer. And "control plasma" herein refers to plasma obtained from these healthy individuals. However, in some applications, for example, when comparing different cancer types, these individuals have other cancer types, and the plasma obtained from these individuals is also designated as a "control".

Typically, the plasma samples used are derived from biological samples from research subjects diagnosed with colorectal cancer. In addition, to be more certain of obtaining data from a "comparator sample," it may be collected from study subjects with known disease states. Biological samples can include body tissues and fluids such as colorectal tissue, serum, blood cells, sputum, and urine. Additionally, biological samples can be obtained from colorectal cancer-characteristic or suspected cases. In addition, samples can be purified from the obtained body tissues and fluids, if necessary, and used as biological samples. According to the present invention, the expression level of the nucleic acid molecule marker is obtained by measuring the biological sample derived from the research object.

The samples used for detection in the in vitro method of the present invention should generally be collected in a clinically acceptable manner, preferably in a manner that protects nucleic acid (especially RNA) or protein. The sample to be analyzed is typically blood. In addition, liver tissue and other types of samples can also be used. Samples can be pooled especially after initial processing. But unpooled samples can also be used.

The term "microRNA" (or "miRNA") as used herein is its ordinary meaning in the art (Bartel, D.P. (2004) Cell 23, 281-292; He, L. and Hannon, G.J. (2004) Nat. Rev. Genet. 5, 522-531). Thus, "microRNA" refers to RNA molecules derived from genomic loci that are processed from transcripts that can form local RNA precursor miRNA structures. Mature miRNAs are typically 20, 21, 22, 23, 24 or 25 nucleotides in length, although other numbers of nucleotides may also be present, such as 18, 19, 26 or 27 nucleotides.

The miRNA coding sequence has the potential to pair with flanking genomic sequences, placing the mature miRNA within a non-perfectly paired RNA duplex (also referred to herein as a stem-loop or hairpin structure or pre-miRNA) that Serves as an intermediate in miRNA processing from longer precursor transcripts. This processing typically occurs through the sequential action of two specific endonucleases called Drosha and Dicer, respectively. Drosha produces miRNA precursors (also referred to herein as "pre-miRNAs") that typically fold into hairpin or stem-loop structures from primary transcripts (also referred to herein as "pri-miRNAs"). From this miRNA precursor, miRNA duplexes are cleaved by Dicer, which contain the mature miRNA in one arm of the hairpin or stem-loop structure and a similarly sized segment in the other arm (commonly referred to as miRNA*). miRNAs are then directed to their target mRNAs to carry out their functions, while miRNAs* are degraded. In addition, miRNAs are typically derived from different genomic segments than predicted protein-coding regions.

The term "miRNA precursor" (or "pre-miRNA" or "pre-miRNA") as used herein refers to the portion of the primary transcript of the miRNA from which the mature miRNA is processed. Typically, pre-miRNAs fold into stable hairpin (ie, duplex) or stem-loop structures. Hairpin structures are typically 50-80 nucleotides in length, preferably 60-70 nucleotides (counting miRNA residues, residues paired with miRNAs, and any intervening segments, but excluding more distal sequences ).

The term "nucleic acid molecule encoding a microRNA sequence" as used herein refers to any nucleic acid molecule encoding a microRNA (miRNA). Thus, the term refers not only to mature miRNAs, but also to corresponding precursor miRNAs and primary miRNA transcripts as described above. In addition, the present invention is not limited to RNA molecules, but also includes corresponding DNA molecules encoding microRNAs, such as DNA molecules produced by reverse transcription of miRNA sequences. A nucleic acid molecule encoding a microRNA sequence of the invention typically encodes a single miRNA sequence (ie, an individual miRNA). However, it is also possible that such a nucleic acid molecule encodes two or more miRNA sequences (i.e. two or more miRNAs), e.g. a transcription unit comprising two or more miRNA sequences.

The term "nucleic acid molecule encoding a microRNA sequence" as used herein is also understood to include a "sense nucleic acid molecule" (i.e. a nucleic acid sequence (5'→3') matching or corresponding to the encoded miRNA (5'→3') sequence molecules) and "antisense nucleic acid molecules" (i.e., the nucleic acid sequence is complementary to the encoded miRNA (5'→3') sequence or in other words matches the reverse complementary sequence (3'→5') of the encoded miRNA sequence molecules). The term "complementary" as used herein refers to the ability of an "antisense" nucleic acid molecule sequence to form base pairs, preferably Watson-Crick base pairs, with a corresponding "sense" nucleic acid molecule sequence (having a sequence complementary to the antisense sequence).

Within the scope of the present invention, two nucleic acid molecules (ie "sense" and "antisense" molecules) may be fully complementary, ie they do not contain any base mismatches and/or additional or missing nucleotides. Alternatively, the two molecules contain one or more base mismatches or differ in their total number of nucleotides (due to additions or deletions). Preferably, a "complementary" nucleic acid molecule comprises at least 10 contiguous nucleotides showing complete complementarity to the sequence comprised in the corresponding "sense" nucleic acid molecule.

Accordingly, the plurality of nucleic acid molecules encoding miRNA sequences contained in the diagnostic kits of the present invention may include one or more "sense nucleic acid molecules" and/or one or more "antisense nucleic acid molecules". Sometimes, diagnostic kits include one or more "sense nucleic acid molecules" (i.e., miRNA sequences themselves) that are believed to make up the repertoire, or at least a subset, of differentially expressed miRNAs (i.e., molecular markers) that are differentially expressed miRNAs are indicative of the presence or predisposition to a particular condition, here colorectal cancer. On the other hand, when the diagnostic kit includes one or more "antisense nucleic acid molecules" (i.e., sequences complementary to miRNA sequences), the molecules may contain one or more molecules suitable for detecting and/or quantifying them in a given sample. Probe molecules (for performing hybridization assays) and/or oligonucleotide primers (eg for reverse transcription or PCR applications) of specific (complementary) miRNA sequences.

A plurality of nucleic acid molecules as defined in the present invention may comprise at least 2, at least 10, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 10000 or at least 100000 nucleic acid molecules, Each molecule encodes a miRNA sequence.

The term "differential expression" as used herein means that the expression level of a particular miRNA in target plasma is altered compared to healthy control plasma, which can be up-regulated (i.e. increased miRNA concentration in target plasma) or down-regulated (i.e. miRNA concentration decreased or disappeared). In other words, the nucleic acid molecule is activated to a higher or lower level in target plasma than in control plasma.

Within the scope of the present invention, a nucleic acid molecule is considered differentially expressed if the corresponding expression levels of the nucleic acid molecule in the target cell and the control cell typically differ by at least 5% or at least 10%, preferably at least 20% or at least 25%, Most preferably at least 30% or at least 50%. Accordingly, the latter value corresponds to the expression level of the given nucleic acid molecule in the target cell being up-regulated by at least 1.3-fold or at least 1.5-fold, respectively, compared to the wild-type control cell, or conversely the expression level in the target cell is down-regulated by at least 0.7-fold or At least 0.5 times.

The term "expression level" as used herein refers to the extent to which a particular miRNA sequence is transcribed from its genomic locus, ie the concentration of the miRNA in one or more analyzed plasmas.

As mentioned above, the term "control plasma" typically refers to (healthy) plasma that does not have the phenotypic characteristics of HCC. However, in some applications, such as when comparing plasmas exhibiting different cancerous or precancerous states, plasma with less severe disease features is typically considered "control plasma".

Determination of expression levels typically follows established standard procedures well known in the art (Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, F.M. et al. (2001) Current Pro-cols in Molecular Biology. Wiley & Sons, Hoboken, NJ). Determination can be performed at the RNA level, for example by Northern blot analysis using miRNA-specific probes, or at the DNA level after reverse transcription (and cloning) of RNA populations, for example by quantitative PCR or real-time PCR techniques. The term "determining" as used herein includes analysis of any nucleic acid molecule encoding the aforementioned microRNA sequence. However, due to the short half-lives of pri-miRNAs and re-mRNAs, only mature miRNA concentrations are typically measured.

In specific embodiments, the expression level obtained in several independent measurements (for example two, three, five or ten measurements) of a given sample and/or in several measurements within a population of target plasma or control plasma Standard values were used for analysis. Standard values can be obtained by any method known in the art. For example, a range of mean ± 2SD (standard deviation) or mean ± 3SD can be used as the standard value.

The difference between the obtained disease or control plasma expression levels can be normalized to the expression levels of further control nucleic acids such as housekeeping genes which are known not to differ according to the disease state of the individual from which the sample was obtained. Exemplary housekeeping genes include β-actin, glyceraldehyde-3-phosphate dehydrogenase, and ribosomal protein P1, among others. In a preferred embodiment, the control nucleic acid is another miRNA known to be stably expressed in the different non-cancerous and (pre-cancerous) states in which the sample was collected.

However, instead of determining expression levels in plasma samples in any experiment, one or more cutoff values for a particular disease phenotype (ie disease state) can also be defined based on experimental evidence and/or prior art data. In this case, lattice expression levels of plasma samples can be determined with stably expressed control miRNAs for normalization. If the calculated "normalized" expression level is above the corresponding defined cut-off value, this finding is indicative of up-regulation of gene expression. Conversely, if the calculated "normalized" expression level is below the corresponding defined cut-off value, this finding is indicative of down-regulation of gene expression.

In the context of the present invention, the term "identifying colorectal cancer and/or differentiating other types of cancer" also includes prediction and likelihood analysis (in the sense of "diagnosis"). The compositions and methods disclosed herein are intended for clinical use to determine treatment modalities, including therapeutic intervention, diagnostic criteria such as disease stage, and disease monitoring and surveillance. According to the present invention, intermediate results for checking the status of objects can be provided. Such intermediate results can be combined with additional information to assist a doctor, nurse or other practitioner in diagnosing the subject with the disease. Alternatively, the invention can be used to detect cancer cells in tissues derived from a subject and provide useful information to physicians for diagnosis. In addition, the present invention is also useful for differentiating colorectal cancer from other types of cancer including hepatocellular carcinoma and lung cancer.

In the present invention, one or more differentially expressed nucleic acid molecules identified together represent a nucleic acid expression signature that is indicative of the presence of colorectal cancer in a plasma sample. The term "expression signature" as used herein refers to a set of nucleic acid molecules (eg, miRNAs), wherein the expression level of each nucleic acid molecule differs between colorectal cancer plasma and healthy controls. Herein, a nucleic acid expression signature also refers to a set of markers and represents a minimum number of (different) nucleic acid molecules each encoding a miRNA sequence that identifies the phenotypic state of an individual.

In one aspect, the present invention relates to a diagnostic kit for determining blood molecular markers of colorectal cancer, the kit comprising multiple nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence. Wherein the one or more nucleic acid molecules are differentially expressed in target plasma and healthy control group, and wherein the differentially expressed markers are derived from tumor-associated or plasma-specific markers, and wherein one or more differentially expressed nucleosides The acid molecules together represent a nucleic acid expression signature indicating the presence of colorectal cancer.

The nucleic acid expression signature defined herein may comprise at least 35 nucleic acid molecules, preferably at least 12 nucleic acid molecules, and particularly preferably at least 6 nucleic acid molecules.

In preferred embodiments, the nucleic acid molecule expression signature comprises at least one nucleic acid molecule encoding a miRNA sequence whose expression is upregulated in one or more plasma compared to one or more control groups, and at least one encoding microRNA sequence A nucleic acid molecule whose expression is downregulated in one or more plasma compared to one or more healthy controls.

As used herein, the term "tumor-derived" or "tumor-derived" refers to a feature that is differentially expressed in the plasma of colorectal cancer patients and controls, and is also differentially expressed in colorectal cancer tissue cells and non-cancer tissue cells.

The colorectal cancer tissue cells used herein refer to colorectal cancer cells excised and collected from subjects diagnosed with colorectal cancer. A non-cancerous tissue cell as used herein generally refers to a (healthy) wild-type cell without the phenotypic characteristics of cancer.

The term "plasma-specific" as used herein refers to differential expression in the plasma of colorectal cancer patients and controls, and no significant difference in colon cancer tissue cells and non-cancer tissue cells.

Typically, the nucleic acid molecules involved in the nucleic acid expression signature are human sequences (hereinafter referred to as "has" (Homo sapiens)).

In a preferred embodiment of the present invention, the nucleic acid expression characteristics include encoding tumor-associated hsa-miR-409-3p (SEQ ID NO: 1), hsa-miR-25 (SEQ ID NO: 2), hsa-miR-93 (SEQ ID NO:3), hsa-miR-96 (SEQ ID NO:4), hsa-miR-301a (SEQ ID NO:5), hsa-miR-342-3p (SEQ ID NO:6), hsa -miR-19b (SEQ ID NO:7), hsa-miR-451 (SEQ ID NO:8), hsa-miR-486-5p (SEQ ID NO:9), hsa-miR-187* (SEQ ID NO: 10), hsa-miR-92a (SEQ ID NO: 11), hsa-miR-19a (SEQ ID NO: 12), hsa-miR-20b (SEQ ID NO: 13), hsa-miR-20a (SEQ ID NO: 14), hsa-miR-139-3p (SEQ ID NO: 15), hsamiR-107 (SEQ ID NO: 16), hsa-miR-17 (SEQ ID NO: 17), hsa-miR-140- Expression of any one or more nucleic acid molecules of 3p (SEQ ID NO: 18), hsa-miR-30e (SEQ ID NO: 19), hsa-miR-185 (SEQ ID NO: 20); encoding plasma specificity hsa-mir-671-3p (SEQ ID NO:21), hsa-mir-16-2* (SEQ ID NO:22), hsa-miR-30c-1* (SEQ ID NO:23), hsa- miR-548c-5p (SEQ ID NO:24), hsa-miR-16 (SEQ ID NO:25), hsa-miR-15a (SEQ ID NO:26), hsa-miR-425 (SEQ ID NO:27 ), hsa-let-7i (SEQ ID NO:28), hsa-miR-363 (SEQ ID NO:29), hsa-miR-15b (SEQ ID NO:30), hsa-miR-101 (SEQ ID NO: :31), hsa-miR-190b (SEQ ID NO: 32) and hsa-miR-130a (SEQ ID NO: 33), hsa-miR-331-3p (SEQ ID NO: 46), hsa-miR-345 ( Expression of any one or more nucleic acid molecules of SEQ ID NO:47); encoding a stable internal stable pair Nucleic acid molecules of hsa-miR-1238 (SEQ ID NO:36) and hsa-miR-1228 (SEQ ID NO:37) according to the same.

In order to normalize the expression levels of nucleic acid molecules (i.e. nucleic acid molecules encoding microRNA sequences contained in the nucleic acid expression profile) in plasma, miRNAhsa-miR-1238 (SEQID NO:44) and hsa-miR- 1228 (SEQ ID NO:45), which is stably expressed in colorectal cancer plasma.

The nucleic acid sequences of the above miRNAs are listed in Table 1.

Table 1

All miRNA sequences disclosed herein have been deposited in the miRBase database (http://microrna.sanger.ac.uk/; see also Griffiths-Jones S. et al. (2008) Nucl. Acids Res. 36, D154- D158).

Particularly preferably, any one or more nucleic acids encoding hsa-miR-409-3p and hsa-mir-671-3p are present in said one or more target plasmas compared to those in one or more control plasmas The expression of molecules was up-regulated, while encoding hsa-miR-25, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR- 451, hsa-miR-486-5p, hsa-miR-187*, hsa-miR-92a, hsa-miR-19a, hsa-miR-20b, hsa-miR-20a, hsa-miR-139-3p, hsa -miR-107, hsa-miR-17, hsa-miR-140-3p, hsa-miR-30e, hsa-miR-185'hsa-mir-16-2*, hsa-miR-30c-1*, hsamiR -548c-5p, hsa-miR-16, hsa-miR-15a, hsa-miR-425, hsa-let-7i, hsa-miR-363, hsa-miR-15b, hsa-miR-101, hsa-miR - The expression of any one or more nucleic acid molecules of 190b, hsa-miR-130a, hsa-miR-331-3p and hsa-miR-345 is down-regulated; hsa-miR-1238 and hsa-miR-1228 have no change.

As used herein, the terms "one or more of the plurality of nucleic acid molecules" and "any one or more human target cell-derived nucleic acid molecules" may relate to any subpopulation of the plurality of nucleic acid molecules, such as any any two kinds, any three kinds, any four kinds, any five kinds, any six kinds, any seven kinds, any eight kinds, any nine kinds, any ten kinds of nucleic acid molecules, each nucleic acid molecule is coded and included in the MicroRNA sequences within the nucleic acid expression characteristics.

In a particularly preferred embodiment, the calculated expression signatures include genes encoding hsa-miR-409-3p/hsa-miR-16-2*, hsa-miR-409-3p/hsa-miR-96, hsa-miR-671- 3p/hsa-miR-548c-5p, hsa-miR-671-3p/hsa-miR-16-2*, hsa-miR-671-3p/hsa-miR-30c-1*, hsa-miR-671- 3p/hsa-miR-342-3p, hsa-miR-671-3p/hsa-miR-96, hsa-miR-671-3p/hsa-miR-301a, hsa-miR-671-3p/hsa-miR- 345, hsa-miR-409-3p/hsa-miR-345, hsa-miR-409/hsa-miR-331-3p, hsa-miR-671-3p/hsa-miR-331-3p, hsa-miR- 671-3p/hsa-miR-25, hsa-miR-409-3p/hsa-miR-93 and hsa-miR-671-3p/hsa-miR-93 any one or a combination of expression of nucleic acid molecules.

The term "nucleic acid combination" as used herein refers to the overall application of at least two nucleic acid expression levels. Relative changes or calculations can preferably be utilized in their entirety through one formula.

In a second aspect, the present invention relates to a diagnostic kit for differentiating colorectal cancer from healthy individuals, hepatocellular carcinoma and lung cancer. The kit comprises a plurality of nucleic acid molecules, each nucleic acid molecule encodes a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules are in target plasma and in one or more healthy individuals, hepatocellular carcinoma and lung cancer differentially expressed, wherein said one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative of the presence of colorectal cancer.

A nucleic acid expression signature as defined herein may comprise at least 23 nucleic acid molecules, preferably at least 18 nucleic acid molecules, and more preferably at least 6 nucleic acid molecules.

In a preferred embodiment, the nucleic acid expression signature comprises at least one nucleic acid molecule encoding a microRNA sequence, whose expression in one or more target plasma is compared with one or more healthy individuals, hepatocellular carcinoma and lung cancer upregulated; and comprising at least one nucleic acid molecule encoding a microRNA sequence whose expression is downregulated in one or more target plasma compared to one or more healthy individuals, hepatocellular carcinoma, and lung cancer.

In a preferred embodiment, the nucleic acid expression features include encoding: tumor-associated features hsa-miR-409-3p (SEQ ID NO: 1), hsa-miR-129-3p (SEQ ID NO: 34), hsa-miR-33b *(SEQ ID NO:35), hsa-miR-7(SEQ ID NO:36), hsa-miR-196b(SEQ ID NO:37), hsa-miR-93(SEQ ID NO:3), hsa- miR-486-5p (SEQ ID NO:9), hsamiR-25 (SEQ ID NO:2), hsa-miR-92a (SEQ ID NO:11), hsa-miR-19b (SEQ ID NO:7); Plasma-specific features hsa-miR-671-3p (SEQ ID NO:21), hsa-miR-16-2* (SEQ ID NO:22), hsa-miR-92b (SEQ ID NO:38), hsa- miR-129* (SEQ ID NO:39), hsa-miR-563 (SEQ ID NO:40), hsa-miR-602 (SEQ ID NO:41), hsamiR-1227 (SEQ ID NO:42), hsa- Any one or more nucleic acid molecules of miR-196a (SEQ ID NO: 43) and internal stability control hsa-miR-1238 (SEQ ID NO: 44) and hsa-miR-1228 (SEQ ID NO: 45).

The nucleic acid sequences of the above miRNAs are listed in Table 2.

Table 2

All miRNA sequences disclosed herein have been deposited in the miRBase database (http://microrna.sanger.ac.uk/; see also Griffiths-Jones S. et al. (2008) Nucl. Acids Res. 36, D154- D158).

In a more preferred embodiment, hsa-miR-409-3p, hsa-mir-671-3p, hsa-miR-33b are encoded in one or more target plasma compared to healthy individuals, hepatocellular carcinoma and lung cancer* , hsa-miR-92b, hsa-miR-149, hsa-miR-129*, hsa-miR-563, hsa-miR-129-3p, hsa-miR-634, hsa-let-7b*, hsa-miR The expression of any one or more nucleic acid molecules of -602 and hsa-miR-1227 is up-regulated, while encoding hsa-miR-16-2*, hsa-miR-7, hsa-miR-196a, hsamiR-196b, hsa- The expression of any one or more nucleic acid molecules of miR-486-5p, hsa-miR-93, hsa-miR-25, hsa-miR-92a and hsa-miR-19b is down-regulated; hsa-miR-1238 and hsa-miR-1238 and hsa- There was no change in miR-1228.

In a more preferred embodiment, the nucleic acid expression signature comprises hsa-miR-33b*/hsa20-miR-196a, hsa-miR-92b/hsa-miR-196a, hsa-miR-563/hsa-miR-196a, hsa-miR-1227/hsa-miR-196a, hsa-miR-129-3p/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-16-2*, hsa-miR-92b /hsa-miR-16-2*, hsa-miR-129*/hsa-miR-196a, hsa-miR-1227/hsa-miR-16-2*, hsa-miR-129-3p/hsa-miR- 196a, hsa-miR-602/hsa-miR-196a, hsa-miR-33b*/hsamiR-16-2*, hsa-miR-563/hsa-miR-16-2*, hsa-miR-129*/ hsa-miR-7, hsa-miR-33b*/hsa25-miR-7, hsa-miR-563/hsa-miR-7, hsa-miR-602/hsa-miR-16-2*, hsa-miR- 129-3p/hsamiR-7, hsa-miR-92b/hsa-miR-7, hsa-miR-1227/hsa-miR-7, hsa-miR-33b*/hsa-miR-196b, hsa-miR-1227 Any one or more nucleic acid combinations of hsa-miR-196b, hsa-miR-92b/hsa-miR-196b and hsa-miR-602/has-miR-7.

In a third aspect, the present invention relates to a method for identifying one or more plasmas exhibiting colorectal cancer, said method comprising: (a) determining the expression levels of a plurality of nucleic acid molecules in said one or more target plasmas , each nucleic acid molecule encodes a microRNA sequence; (b) determines the expression level of said plurality of nucleic acid molecules in one or more healthy control plasma; (c) by comparing the obtained in steps (a) and (b) Respective expression levels, one or more nucleic acid molecules differentially expressed in the target plasma and control plasma are identified from the plurality of nucleic acid molecules, wherein the differentially expressed one or more nucleic acid molecules together represent A defined signature that is indicative of the presence of colorectal cancer.

In a preferred embodiment of the present invention, the method includes (a) determining the expression level of the combination of nucleic acid molecules in one or more target plasma, each nucleic acid molecule is encoded microRNA sequence, and calculated with a specific formula; (b ) determining the expression level of the combination of nucleic acid molecules in one or more healthy control plasma, and calculating with a specific formula; and (c) comparing the respective expression levels obtained in steps (a) and (b) to determine Identifying differences in said combination in said one or more target plasmas, wherein the one or more differentially expressed combinations represent features indicative of the presence of colorectal cancer.

In a more preferred embodiment, the method is further used to differentiate colorectal cancer from healthy individuals, hepatocellular carcinoma and lung cancer.

In a fourth aspect, the present invention relates to a method for monitoring treatment of colorectal cancer, the method comprising: (a) identifying nucleic acid expression signatures in one or more target plasmas by using the methods defined herein; and (b) in Monitoring the expression of one or more nucleic acid molecules encoding microRNA sequences included in the nucleic acid expression signature in the blood in such a way that the expression of the nucleic acid molecules whose expression in plasma was up-regulated before treatment The expression of nucleic acid molecules whose expression in plasma was downregulated before treatment is downregulated after treatment is upregulated after treatment.

As used herein, the term "altering the expression of a nucleic acid molecule encoding a miRNA sequence" refers to any manipulation of a specific nucleic acid molecule to result in an altered expression level of said molecule, i.e. compared to the expression of a "wild type" (i.e. unaltered control) Compared to produce different amounts of the corresponding miRNA. As used herein, the term "different amount" includes both higher and lower amounts compared to an unaltered control. In other words, manipulation as defined herein may be up-regulation (i.e. activation) or down-regulation (i.e. repression) of the expression (i.e. in particular transcription) of a nucleic acid molecule

In a fifth aspect, the present invention relates to a method for preventing or treating colorectal cancer, said method comprising: (a) identifying nucleic acid expression characteristics in plasma using the methods defined herein; and (b) altering said nucleic acid in blood The expression profile comprises the expression of one or more nucleic acid molecules encoding microRNA sequences, said alteration being carried out in such a way that the expression of nucleic acid molecules whose expression is up-regulated in blood is down-regulated, whereas the expression of nucleic acid molecules whose expression is up-regulated in blood Expression of a downregulated nucleic acid molecule is upregulated.

In the present invention, the expression of one or more nucleic acid molecules encoding microRNA sequences contained in the nucleic acid expression profile is modified in such a way that its expression is upregulated in the one or more target plasma Expression of the molecule is downregulated and expression of the nucleic acid molecule whose expression is downregulated in the one or more target plasma is upregulated. In other words, the modification of the expression of a specific nucleic acid molecule encoding a miRNA sequence occurs in an anti-cyclical manner to the regulation of said molecule in plasma of said one or more cancer targets, so that in said one or more Interfering with the "overactivity" of upregulated molecules and/or restoring the "deficit activity" of downregulated molecules in plasma of one or more of the targets.

In a preferred embodiment of the method of the present invention, down-regulating the expression of the nucleic acid molecule comprises introducing into the patient a nucleic acid molecule encoding a sequence complementary to the microRNA sequence encoded by the down-regulated nucleic acid molecule.

The term "introducing into blood" as used herein refers to any manipulation that results in the transfer of one or more nucleic acid molecules into the blood. Examples of such techniques include injection, digestion and other techniques well known in the art.

The term "complementary sequence" as used herein refers to a "complementary" nucleic acid molecule (also referred to herein as an "antisense nucleic acid molecule") introduced into one or more cells capable of forming bases with an upregulated endogenous "sense" nucleic acid molecule Yes, Watson-Crick base pairs are preferred.

Two nucleic acid molecules (ie, "sense" and "antisense" molecules) can be perfectly complementary, ie, they do not contain any base mismatches and/or added or deleted nucleotides. In other embodiments, the two molecules contain one or more base mismatches or differ in their total number of nucleotides (due to additions or deletions). In additional embodiments, a "complementary" nucleic acid molecule comprises a stretch of at least ten contiguous nucleotides that is completely complementary to the sequence comprised in the upregulated "sense" nucleic acid molecule.

A "complementary" nucleic acid molecule (i.e., a nucleic acid molecule encoding a nucleic acid sequence complementary to the microRNA sequence encoded by the down-regulated nucleic acid molecule) may be a naturally occurring DNA- or RNA molecule or contain in its sequence one or more of the same type or a Or a synthetic nucleic acid molecule of many different types of modified nucleotides.

For example, it is possible that such a nucleic acid molecule comprises at least one ribonucleotide backbone unit and at least one deoxyribonucleotide backbone unit. In addition, the nucleic acid molecule may contain one or more RNA backbone modifications as 2'-O-methyl groups or 2'-O-methoxy groups (also known as 2'-O-methylation) , which prevents nuclease degradation in culture medium and importantly also prevents inner core dissociation of RNA-induced silencing complex nucleases, leading to irreversible inhibition of miRNAs. Another possible modification (which is functionally equivalent to 2'-O-methylation) involves locked nucleic acid (LNA), which stands for nucleic acid analog containing one or more LNA nucleomonomers, which are present in RNA Simulated sugar conformation with locked bicyclic furanose units (Orom, U.A. et al. (2006) Gene 372, 137-141).

Another class of miRNA expression silenced genes was recently developed. These chemically engineered oligonucleotides, called "antagomirs", are single-stranded 23 nucleotide RNA molecules conjugated to cholesterol (Krutzfeldt, J. et al. (2005) Nature 438, 685-689). As an alternative to such chemically modified oligonucleotides, microRNA inhibitors that can be expressed in cells are produced as RNA from transgenes. These competitive inhibitors, termed "microRNA sponges," are transcripts expressed from strong promoters that contain multiple tandem binding sites for the microRNA of interest (Ebert, M.S. et al. (2007) Nat. Methods 4, 721-726).

In a particularly preferred embodiment of the method of the invention, the one or more nucleic acid molecule encodings whose expression is down-regulated relative to the expression profile are selected from the group consisting of microRNA sequences: hsa-miR-25, hsa-miR-93 , hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsamiR-486-5p, hsa-miR-187*, hsa-miR- 92a, hsa-miR-19a, hsa-miR-20b, hsa-miR-20a, hsa-miR-139-3p, hsa-miR-107, hsa-miR-17, hsa-miR-140-3p, hsa- miR-30e, hsa-miR-185, hsa-miR-16-2*, hsa-miR-30c-1*, hsa-miR-548c-5p, hsa-miR-16, hsa-miR-15a, hsa- miR-425, hsa-let-7i, hsa-miR-363, hsa-miR-15b, hsa-miR-101, hsa-miR-190b, hsa-miR-130a, hsa-miR-7, hsa-miR- 196b and hsa-miR-196a, as mentioned above may be indications for colorectal cancer.

In another preferred embodiment of the method of the present invention, up-regulating the expression of the nucleic acid molecule comprises introducing into the blood a nucleic acid molecule encoding a microRNA sequence encoded by the up-regulated nucleic acid molecule. In other words, upregulation of expression of a nucleic acid molecule encoding a miRNA sequence is achieved by introducing another copy of said miRNA sequence (ie, an additional "sense" nucleic acid molecule) into said one or more cells. The "sense" nucleic acid molecules introduced into one or more target cells may contain the same modifications as the "antisense" nucleic acid molecules described above.

In a particularly preferred embodiment of the method of the present invention, the one or more nucleic acid molecule encodings whose expression is up-regulated relative to the expression characteristics are microRNA sequences selected from the group consisting of: hsa-miR-409-3p, hsa-miR -671-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-92b, hsa-miR-129*, hsa-miR-563, hsa-miR-602 and hsa-miR-1227 , as mentioned above may be an indication for colorectal cancer.

"Sense" and/or "antisense" nucleic acid molecules introduced into the blood to modify the expression of one or more nucleic acid molecules encoding microRNA sequences contained in a nucleic acid expression signature can be operably linked with regulatory sequences so that all expression of the nucleotide sequence.

To elucidate any potential association of miRNAs identified in cancerous or precancerous samples, preparatory functional analyzes can be performed with regard to the identification of mRNA target sequences to which the miRNAs can bind. Based on the discovery that miRNAs can be involved in both tumor suppression and tumorigenesis (Esquela-Kerscher, A. and Slack, F.J (2006) supra; Calin, G.A. and Croce, C.M. (2007) supra; Blenkiron, C. and Miska , E.A. (2007) as above), it can be speculated that the mRNA target sites of this miRNA include tumor suppressor genes as well as oncogenes.

A nucleic acid molecule is said to be an "expressing nucleic acid molecule" or capable of "Causing expression of a nucleotide sequence". Operably linked is one in which the regulatory sequence element and the expressed sequence (and/or reciprocally expressed sequences) are linked in such a way that the gene is expressed.

The exact nature of the regulatory regions necessary for gene expression can vary in different species, but generally these regions contain a promoter, which in prokaryotes contains two promoters, the DNA element directing the initiation of transcription and the A DNA element that signals translation initiation. Such promoter regions usually include 5' noncoding regions involved in initiation of transcription and translation, such as the -35/-10 box and Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequence and 5'-capping element. These regions may also include enhancer or repressor elements as well as translation signals and leader sequences to target the native polypeptide to specific compartments of the host cell. In addition, the 3' non-coding sequences may contain regulatory elements involved in transcription termination, polyadenylation, and the like. However, if these termination sequences do not function satisfactorily in a particular host cell, they can be replaced with signals that are functional in that cell.

Furthermore, the expression of a nucleic acid molecule as defined herein may also be influenced eg by the presence of modified nucleotides (as described above). For example, locked nucleic acid (LNA) monomers are thought to increase the functional half-life of miRNAs in vivo by enhancing resistance to degradation and by stabilizing the miRNA-target duplex structure critical for silencing activity (see, e.g., Naguibneva, I. et al. (2006) Biomed. Pharmacother 60, 633-638).

Thus, the nucleic acid molecules of the invention introduced into the blood provided may comprise regulatory sequences, preferably promoter sequences, and optionally also transcription termination sequences. The promoter may allow constitutive or inducible gene expression. Suitable promoters include the E. coli lacUV5 and tet (tetracycline responsive) promoters, the T7 promoter and the SV40 promoter or CMV promoter.

The nucleic acid molecules of the invention may also be contained in vectors or other cloning vehicles such as plasmids, phagemids, phages, cosmids or artificial chromosomes. In a preferred embodiment, said nucleic acid molecule is comprised in a vector, in particular an expression vector. Such expression vectors may comprise, in addition to the aforementioned regulatory sequences and nucleic acid sequences encoding the genetic constructs as defined in the present invention, replication and control sequences derived from species compatible with the host used for expression and conferring a selectable expression on the transfected cells. type selection markers. Many suitable vectors are known in the art and are commercially available, such as pSUPER and pSUPERIOR.

In a sixth aspect, the present invention relates to a pharmaceutical composition for preventing and/or treating colorectal cancer in blood, said composition comprising one or more nucleic acid molecules, each nucleic acid molecule coding sequence, said sequence being as As defined herein, the microRNA sequence encoded by the nucleic acid molecule whose expression is upregulated in the blood plasma from a colorectal cancer patient is at least partially complementary, and/or the sequence corresponds to the microRNA sequence as defined herein in a blood plasma from a colorectal cancer patient. A microRNA sequence encoded by a nucleic acid molecule whose expression is downregulated in plasma.

Finally, in the seventh aspect, the present invention relates to the use of the pharmaceutical composition in the preparation of a medicament for preventing and/or treating hepatocellular carcinoma.

Suitable pharmaceutical compositions within the scope of the present invention include those suitable for oral, rectal, nasal, whole (including buccal, and jaw), peritoneal, and parenteral (including intramuscular, subcutaneous, and intravenous) procedures, or by inhalation. Or blown into the implementation. Actions can be local or systemic. Preferably, the procedure is accomplished by oral or intravenous routes. Formulations can be packaged in different dosage units.

The pharmaceutical compositions of the present invention include any pharmaceutical dosage form established in the art, such as capsules, microcapsules, cachets, pills, tablets, powders, pellets, multiparticulate formulations (such as beads, granules or crystals) , aerosols, sprays, foams, solutions, dispersions, tinctures, syrups, elixirs, suspensions, water-in-oil emulsions such as ointments, and oil-in-water emulsions such as creams, lotions and balms.

The aforementioned ("sense" and "antisense") nucleic acid molecules can be formulated into pharmaceutical compositions using pharmacologically acceptable ingredients and established manufacturing methods (Gennaro, A.L. and Gennaro, A.R. (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA; Crowder, T.M. et al. (2003) A Guide to Pharmaceutical Particulate Science. Interpharm/CRC, Boca Raton, FL; Niazi, S.K. (2004) Handbook of Pharmaceutical Manufacturing Formulations, CRC Press, Boca Raton, FL).

For the preparation of pharmaceutical compositions, pharmaceutically inert, inorganic or organic excipients (ie carriers) may be used. For the preparation of, for example, pills, tablets, capsules or granules, it is possible to use, for example, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyols, natural or hardened oils. Suitable excipients for the manufacture of solutions, suspensions, emulsions, aerosol mixtures or powders for reconstitution into solutions or aerosol mixtures prior to use include water, alcohols, glycerol, polyols and suitable mixtures thereof and vegetable oil.

The pharmaceutical composition may also contain additives, such as fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and further solvents or solubilizers or substances that achieve a storage effect. The latter is understood to mean that nucleic acid molecules can be incorporated into slow or sustained release or targeted delivery systems such as liposomes, nanoparticles and microcapsules.

In order to target most tissues in the body, clinically feasible non-invasive strategies are required to target such pharmaceutical compositions as defined herein to cells. In the past, several approaches have achieved significant therapeutic benefit by intravenously injecting reasonable doses of siRNA into mice and primates without significant limiting toxicity.

One approach involves covalently coupling the passenger strand of miRNA (miRNA* strand) to cholesterol or its derivatives/conjugates to facilitate uptake through ubiquitously expressed cell surface LDL receptors (Soutschek, J. et al. (2004) Nature 432, 173-178). Alternatively, unconjugated PBS-formulated locked nucleic acid modified oligonucleotides (LNA-antimiR) can be used for systemic delivery (Elmen, J. et al. (2008) Nature 452, 896-899). Another method of miRNA delivery involves encapsulation of miRNA into specific liposomes using polyethylene glycol to reduce uptake by clearing cells and enhance circulation time. These specific nucleic acid particles (stabilized nucleic acid-lipid particles or SNALPs) efficiently deliver miRNAs to the liver (and not to other organs (described by Zimmermann, T.S. et al. (2006) Nature 441, 111-114)). In recent years, a new class of lipid-like delivery molecules called lipidoids (synthesized based on the conjugation addition of alkylacrylates or alkyl-acrylamides to primary or secondary amines) has been described as delivery agents for RNAi therapeutics (Akinc, A. et al. (2008) Nat. Biotechnol. 26, 561-569).

A further cell-specific targeting strategy involves mixing miRNAs with a fusion protein consisting of targeting antibody fragments linked to protamine, which nucleates DNA in sperm and passes charge Basic protein that binds miRNA (Song, E. et al. (2005) Nat. Biotechnol. 23, 709-717). Many modifications and variations of the basic delivery method described above have been developed recently. These techniques are known in the art and are reviewed, for example, in de Fougerolles, A. et al. (2007) Nat. Rev. Drug Discov. 6, 443-453; Kim, D.H. and Rossi, J.J. (2007) Nat. Genet. 8, 173 -184).

The present invention is further described by the accompanying drawings and the following examples, which are only for the purpose of illustrating specific embodiments of the present invention and should not be construed as limiting the scope of the present invention in any way.

Example

Embodiment 1: sample collection and preparation

Sixty-nine colorectal cancer tissues and 69 matched normal colorectal cancer tissue samples were obtained from surgery. Surgical samples were snap frozen in liquid nitrogen at or shortly after collection, and samples were stored at -80°C.

Patient information (age, gender, imaging data, treatment, other medication status, family history, and the like) was sourced from hospital databases to allow matching of different samples collected. Pathological follow-up (e.g., histological analysis by hematoxylin and eosin staining (H&E)) is used to significantly determine the disease status of a given sample (i.e., control, precancerous (e.g., colorectal adenoma), early malignant lesion (e.g., colorectal adenoma), cancer)), and to ensure consistent classification of samples.

Laser capture microdissection was optionally performed on each cancer sample to specifically isolate the tumor cell population (approximately 200,000 cells). Briefly, a clear transfer film is applied to the surface of a tissue section or sample. Under a microscope, thin tissue sections mounted on glass slides are viewed and cell populations are identified for isolation. When the selected cell is in the center of the observed field of view, a near IR laser diode integral with the microscope optics is activated. A pulsed laser beam activates spots on the transferred membrane, causing the membrane to fuse with the underlying selected cells. The transferred membrane with bound cells is then stripped from the thin tissue section (for review see e.g. Emmert-Buck, MR et al. (1996). Science 274, 998-1001; Espina, V. et al. (2007) Expert Rev. Mol . Diagn. 7, 647-657). Cryosections were prepared essentially as directed by the manufacturer and the capture step was performed using a laser capture microscope (Arcturus Veritas ^™ Laser Capture Microdissection Instrument (Molecular Devices, Inc., Sunnyvale, CA, USA).

miRNA populations were isolated using the mirVana ^™ miRNA Isolation Kit (Ambion, Inc., Austin, TX, USA) according to the manufacturer's instructions. Concentrations were quantified by NanoDrop 1000 spectrophotometer. (NanoDrop Technologies, Waltham, MA). Quality control of RNA was achieved by 2100Bioanalyzer using RNA 6000Pico LabChip kit (Agilent Technologies, Santa Clara, CA).

Example 2: Analysis of miRNA expression profiles in tissue samples

Qualitative analysis of (differentially) expressed miRNAs in specific samples was optionally performed using the Agilent miRNA microarray platform (Agilent Technologies, Santa Clara, CA, USA). The microarray included 723 human miRNA probes obtained from the Sanger database v.10.1. Total RNA (100 ng) derived from each 138LCM-selected colorectal cancer sample was used as a marker pool by Cy3 population. Microarray image results were obtained by XDR scanning (PMT100, PMT5). Labeling and hybridization were performed according to the steps of the Agilent miRNA microarray system.

Raw data obtained for single-color (CY3) hybridizations were normalized by applying the Quantile method and using GeneSpring GX10 software known in the art (Agilent Technologies, Santa Clara, CA, USA). Unpaired t-test (p-value <0.01) was used to identify differentially expressed miRNAs in colorectal cancer and matched normal control tissues after Fisher's assay (F-test).

An independent experiment on 138 tissue samples was performed for each measurement and the determined miRNA expression levels represent the average of the individual data obtained.

Example 3: Collection and preparation of plasma samples

The main method steps for determining the expression characteristics of one or more nucleic acids in colorectal cancer target plasma are shown in FIG. 1 .

114 blood samples from cancer patients and healthy individuals were collected from Zhongshan and Huashan Hospitals in Shanghai in 2008 and 2009. The basic characteristics of the blood samples used in the present invention are shown in Figure 3. All samples obtained from patients were obtained surgically. Patient data (age, gender, imaging data, treatment methods, other medical conditions, family history, etc.) were sourced from hospital databases. The histopathological classification of the tumor was obtained independently by three pathologists according to the WHO tumor classification system.

Table 3: Basic characteristics of blood samples

Genome-wide miRNA analysis Number of people comparison normal twenty three Hepatocellular carcinoma twenty four lung cancer 36 bowel cancer Dukes'A carcinoma 6 Dukes' B carcinoma 9 Dukes'C carcinoma 9 Dukes'D cancer 7  Quantitative RT-PCR analysis normal 54 bowel cancer 54 Total 222

Peripheral blood (2ml) was transferred to EDTA tubes. Within two hours, the EDTA tubes were centrifuged at 820 g for 10 min. A 1-ml aliquot of plasma was then transferred to a 1.5-ml vial and centrifuged at 16,000 g for 10 min to pellet any remaining cellular debris. The supernatant was then transferred to a new tube and stored at -80°C.

Total RNA was extracted from plasma using the mirVana PARIS miRNA extraction kit (Ambion, Austin, TX) according to the manufacturer's instructions. Concentrations were quantified by NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Waltham, MA). RNA quality control was achieved with the 2100 Bioanalyzer using the RNA 6000 Pico LabChip kit (Agilent Technologies, Santa Clara, CA).

Example 4: Analysis of miRNA expression profiles in plasma samples

Qualitative analysis of miRNA(f differences) in specific plasma samples can optionally use the Agilent miRNA microarray platform (Agilent Technologies, Santa Clara, CA, USA). The microarray included probes for 723 human miRNAs obtained from the Sanger database v.10.1. Total RNA (100 ng) was derived from each of 114 plasma samples and was used as a marker pool by Cy3 population. Microarray impact results were obtained by XDR scanning (PMT100, PMT5). Labeling and hybridization were performed according to the steps of the Agilent miRNA microarray system.

For data analysis, raw data from single-color (CY3) hybridizations were normalized by using a stable internal miRNA control (has-miR-1238). Unpaired t-tests were used to identify differentially expressed miRNAs in colorectal cancer and healthy individuals and/or colorectal cancer and controls (healthy individuals, hepatocellular carcinoma, and lung cancer) after Fisher's assay (F-test).

To determine the specificity and sensitivity of individual miRNAs as diagnostic biomarkers, MedCalc software was used to perform receiver operating characteristic (ROC) curve analysis of individual miRNAs versus healthy individuals and/or controls (healthy individuals, hepatocellular carcinoma, and lung cancer). 95% confidence intervals were used to determine significance.

To assess the differential expression of a specific miRNA in early-stage HCC or metastatic HCC compared to healthy individuals or controls, the following criteria were used:

i) p-value (likelihood value) <= 0.01 fold change > 2

ii) AUC (accuracy as a diagnostic biomarker) AUC>0.700

Where both criteria were fulfilled, miRNAs were considered to be differentially expressed in colorectal cancer relative to healthy individuals and/or controls.

An independent experiment was performed on 114 plasma samples to obtain the individual measurements, and the miRNA expression levels represent the average of the individual data obtained.

The experimental data of differentially expressed miRNAs obtained from CRC relative to healthy controls are summarized in the following Tables 4-6. Table 4 lists miRNAs that are significantly differentially expressed in tissue and plasma as exhibited by CRC relative to control tissue and healthy control plasma. Table 5 summarizes miRNAs differentially expressed in plasma only in colorectal cancer patients compared to healthy individuals. Table 6 lists the best miRNA signature combinations in the plasma of CRC cases. Column "t" refers to colorectal cancer tissue, "n" refers to matched normal control tissue, "p" refers to patient, and "h" refers to healthy control. Particularly preferred miRNAs (SEQ ID NO: shown in Table 4 for SEQ ID NO:8; and Table 5 for SEQ ID NO:21 versus SEQ ID NO:24) are bolded.

Table 4: Tumor-associated miRNA signatures in colorectal cancer plasma

Table 5: Plasma-specific miRNA signatures in colorectal cancer plasma

Table 6: Combinatorial miRNA signatures in colorectal cancer plasma

Preferred expression signature data for distinguishing colorectal cancer from healthy individuals, hepatocellular carcinoma and lung cancer in the second aspect are listed in Tables 7-9 below. Table 7 lists colorectal cancer tumor-associated miRNA features; Table 8 shows plasma-specific miRNA features; Table 9 shows the best combination of colorectal cancer miRNA features, best combination (hsa-miR-33b*/hsa-miR-196a) 85% accuracy as a diagnostic biomarker for distinguishing CRC from healthy individuals, hepatocellular carcinoma and lung cancer. The "t" column is colorectal cancer tissue, the "n" column is matched normal control tissue, the "CRC" column is colorectal cancer plasma, and the "control" column is plasma obtained from healthy individuals, hepatocellular carcinoma and lung cancer patients. Preferred miRNA (SEQ ID NO:1; SEQ ID NO:34-37; SEQ ID NO:3 is shown in Table 7, SEQ ID NO:21, SEQ ID NO:38-42 is shown in Table 8, SEQ ID NO:35 and SEQ ID NO:43 are shown in Table 9) are the blackened part.

Table 7: Tumor-associated miRNA signatures distinguishing colorectal cancer from healthy controls, hepatocellular carcinoma and lung cancer

Table 8: Plasma-specific miRNA signatures distinguishing colorectal cancer from healthy controls, hepatocellular carcinoma and lung cancer

Table 9: Combinatorial miRNA signatures distinguishing colorectal cancer from healthy controls, hepatocellular carcinoma and lung cancer

Example 5: Verification of microarray data

To validate (and/or quantify) the miRNA expression data obtained, an established real-time quantitative RT-PCR using the TaqMan MicroRNA assay (Applied Biosystems, Foster City, CA, USA) was used according to the manufacturer's instructions. Briefly, reverse transcription (RT) was achieved using the Taqman microRNART kit according to the instructions of the biological system used. 10ng total RNA reverse transcription solution mix is 15ul, including 1X reverse transcription buffer, 1X RT primer, 1nM dNTP, 4U RNase inhibitor and 50U MultiScribe reverse transcriptase. Then the RT solution was operated in a PCR instrument by thermal program 16°C, 30min; 42°C, 30min; 85°C, 5min (Thermal cycler alpha engine, Bio-rad). Quantitative PCR was performed with TaqMan Universal PCR Master Mix Kit and Taqman MicroRNA Assay Kit according to the instructions of the biological system used. The PCR amplification conditions for 2ul RT products are: 1XTaqMan Universal PCR Master Mix, without AmpErase UNG, 1X TaqMan MicroRNA assay mix. Each step of translation goes through three cycles. The operating condition of real-time PCR is Roch Light Cycling 480 instrument, the program is to start heating at 96°C for 5min; then 95°C for 15s to go through 45 or 50 cycles; 60°C for 60s. The Cp value is calculated by the second derivation method of LC480 software. Then the miRNAs were absolutely quantified by the CP value of the standard sample, and the CP value of each miRNA was normalized to the internal stability control has-miR-1228.

The platform comparison experiment data of 8 miRNA combinations and 7 miRNAS obtained from plasma samples of 54 healthy individuals and 54 cases of colorectal cancer are listed in Table 10. Overall, the array fold difference between patients and healthy controls was greater than that of quantitative RTPCR. Modulation (ie, upregulation or downregulation) obtained by array results correlates strongly with quantitative RT-PCR results. The results show that miRNA signatures discovered by Agilent miRNA microarrays are highly reliable.

Table 10: Platform comparison of colorectal cancer patient plasma and healthy individual plasma expression profiles

The obtained results indicate an overall highly specific regulation of miRNA expression in colorectal cancer. Thus, here specific miRNA subsets each represent a unique miRNA expression signature, as colorectal expression profiles not only determine cancer status but also differentiate from hepatocellular and lung cancers.

The determination of miRNA expression characteristics of the present invention provides a unique molecular marker for blood screening, detection and diagnosis of colorectal cancer. In addition, expression signatures can be used to monitor treatment response and guide treatment strategies in colorectal cancer patients. In addition, expression signatures can also be used to develop anti-colorectal cancer drugs.

The invention exemplified herein can suitably be practiced in the absence of any element, limitation, specifically disclosed herein. Thus, for example, the terms "comprises," "including," "containing," etc., shall have a broad meaning and are not limiting. In addition, the terms and expressions used herein are used to describe the present invention in a non-limiting sense, and there is no intention to use these terms and expressions to exclude any of the features and descriptions shown or equivalents thereof, but it should be recognized that in the claimed Various modifications can be made within the scope of the present invention. Therefore, it is to be understood that although the invention has been particularly disclosed by way of embodiments and optional features, modifications and changes of the invention may be made by those skilled in the art and such modifications and changes are considered to be within the scope of the invention.

The invention has been described broadly and generically herein. Every narrower generic concept and subgeneric collection that falls within the generic description also forms a part of this invention. This includes a generic description of the invention with a proviso or negative limitation removing any subject matter from the generic, regardless of whether the removed subject matter is specifically recited herein.

Other embodiments are within the scope of the following claims. In addition, when features or aspects of the invention are described in terms of Markush groups, those skilled in the art will appreciate that the invention is also described in terms of any individual member or subgroup of members of the Markush group.

Claims (16)

1., for differentiating that one or more shows the diagnostic kit of the blood Middle molecule marker of the target plasma of colorectal cancer, described test kit comprises the probe molecule of multiple nucleic acids molecule, often kind of nucleic acid molecule encoding microrna sequences,

One or more differential expression in target plasma and in one or more contrast blood plasma of wherein said multiple nucleic acids molecule,

The nucleic acid molecule of one or more differential expression wherein said represents expression of nucleic acid feature together, described expression of nucleic acid feature is the indication that colorectal cancer exists, wherein said expression of nucleic acid feature comprises coding hsa-miR-409-3p/hsa-miR-16-2*, hsa-miR-409-3p/hsa-miR-96, hsa-miR-671-3p/hsa-miR-548c-5p, hsa-miR-671-3p/hsa-miR-16-2*, hsa-miR-671-3p/hsa-miR-30c-1*, hsa-miR-671-3p/hsa-miR-342-3p, hsa-miR-671-3p/hsa-miR-96, hsa-miR-671-3p/hsa-miR-301a, hsa-miR-671-3p/hsa-miR-345, hsa-miR-409-3p/hsa-miR-345, hsa-miR-409/hsa-miR-331-3p, hsa-miR-671-3p/hsa-miR-331-3p, hsa-miR-671-3p/hsa-miR-25, any one or the multiple nucleic acids molecular combinations of hsa-miR-409-3p/hsa-miR-93 and hsa-miR-671-3p/hsa-miR-93.

2. the test kit of claim 1, wherein said expression of nucleic acid feature can comprise at least 35 kinds of nucleic acid molecule.

3. the test kit of claim 1, wherein said expression of nucleic acid feature can comprise at least 12 kinds of nucleic acid molecule.

4. the test kit of claim 1, wherein said expression of nucleic acid feature can comprise at least 6 kinds of nucleic acid molecule.

5. the test kit of claim 1, wherein compared with one or more normal healthy controls, in one or more target plasma described: the expression of any one or the multiple nucleic acids molecule of coding hsa-miR-409-3p and hsa-mir-671-3p is raised; And any one or the multiple nucleic acids developed by molecule of encode hsa-miR-25, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-mir-16-2*, hsa-miR-30c-1*, hsa-miR-548c-5p, has-miR-331-3p and hsa-miR-345 are lowered.

6. the test kit of claim 1, is further used for colorectal cancer and healthy individuals, hepatocellular carcinoma and lung cancer to differentiate.

7. the test kit of claim 6, wherein said expression of nucleic acid feature can comprise at least 23 kinds of nucleic acid molecule.

8. the test kit of claim 6, wherein said expression of nucleic acid feature can comprise at least 18 kinds of nucleic acid molecule.

9. the test kit of claim 6, wherein said expression of nucleic acid feature can comprise at least 6 kinds of nucleic acid molecule.

10. the test kit of claim 6, wherein said expression of nucleic acid feature comprises any one or Multi-encoding: tumour correlated characteristic hsa-miR-409-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-7, hsa-miR-196b, hsa-miR-93, hsa-miR-486-5p, hsa-miR-25, hsa-miR-92a, hsamiR-19b; The nucleic acid molecule of plasma specific feature hsa-miR-671-3p and hsa-miR-16-2*, has-miR-92b, hsa-miR-129*, hsa-miR-563, hsa-miR-602, hsa-miR-1227, hsa-miR-196a and internal stability contrast hsa-miR-1238 and hsa-miR-1228.

The test kit of 11. claims 10, wherein compare with lung cancer with one or more healthy individuals, hepatocellular carcinoma, in one or more target plasma described, any one or the multiple nucleic acids developed by molecule of coding hsa-miR-409-3p, hsa-mir-671-3p, hsa-miR-33b*, hsa-miR-92b, hsa-miR-149, hsa-miR-129*, hsa-miR-563, hsa-miR-129-3p, hsa-miR-634, hsa-let-7b*, hsa-miR-602 and hsa-miR-1227 are raised; And any one or the multiple nucleic acids developed by molecule of encode hsa-miR-16-2*, hsa-miR-7, hsa-miR-196a, hsa-miR-196b, hsa-miR-486-5p, hsa-miR-93, hsa-miR-25, hsa-miR-92a and hsa-miR-19b are lowered; Hsa-miR-1238 and hsa-miR-1228 does not change.

The test kit of 12. any one of claim 6-11, wherein said expression of nucleic acid feature comprises coding: hsa-miR-33b*/hsa-miR-196a, hsa-miR-92b/hsa-miR-196a, hsa-miR-563/hsa-miR-196a, hsa-miR-1227/hsa-miR-196a, hsa-miR-129-3p/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-16-2*, hsa-miR-92b/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-196a, hsa-miR-1227/hsa-miR-16-2*, hsa-miR-129-3p/hsa-miR-196a, hsa-miR-602/hsa-miR-196a, hsa-miR-33b*/hsa-miR-16-2*, hsa-miR-563/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-7, hsa-miR-33b*/hsa-miR-7, hsa-miR-563/hsa-miR-7, hsa-miR-602/hsa-miR-16-2*, hsa-miR-129-3p/hsa-miR-7, hsa-miR-92b/hsamiR-7, hsa-miR-1227/hsa-miR-7, hsa-miR-33b*/hsa-miR-196b, hsa-miR-1227/hsa-miR-196b, any one or the multiple nucleic acids combination of hsa-miR-92b/hsa-miR-196b and hsa-miR-602/hsa-miR-7.

The test kit of 13. any one of claim 6-12 is for the preparation of the purposes differentiating by the following method to show in the composition of one or more target plasma of colorectal cancer, and described method comprises:

A () determines the expression level of multiple nucleic acids molecule in one or more target plasma described, often kind of nucleic acid molecule encoding microrna sequences;

B () determines the expression level of described multiple nucleic acids molecule in one or more normal healthy controls blood plasma; And

C respective expression level that () is obtained in step (a) and (b) by contrast, one or more nucleic acid molecule of differential expression in described target plasma and contrast blood plasma is identified from described multiple nucleic acids molecule, one or more nucleic acid molecule of wherein said differential expression represent together as any one of claim 1-12 the expression of nucleic acid feature that defines, described expression of nucleic acid feature is the indication that colorectal cancer exists.

The purposes of 14. claims 13, wherein said method is further used for colorectal cancer and healthy individuals, hepatocellular carcinoma and lung cancer to differentiate.

15. the test kit of any one of claim 1-12 is for the preparation of the purposes of monitoring by the following method in the composition for the treatment of of colorectal cancer, described method comprises:

A () differentiates expression of nucleic acid feature by using method defined herein in one or more target plasma; And

B () monitors the expression of one or more nucleic acid molecule of the coding microrna sequences that described expression of nucleic acid feature comprises in blood, described monitoring is carried out in such a way, namely the expression in its blood plasma is lowered after the treatment by the expression of the nucleic acid molecule raised before the treatment, and the expression in its blood plasma is raised after the treatment by the expression of the nucleic acid molecule lowered before the treatment.

The test kit of 16. any one of claim 1-12 is for the preparation of the purposes in the test kit of prevention or treatment colorectal cancer by the following method, and described method comprises:

A () differentiates expression of nucleic acid feature in blood by using the method described in claim 13 or 14; And

B () changes the expression of one or more nucleic acid molecule of the coding microrna sequences that described expression of nucleic acid feature comprises in blood, described change is carried out in such a way, namely its expression is lowered by the expression of the nucleic acid molecule raised in blood, and its expression is raised by the expression of the nucleic acid molecule lowered in blood.