CN118127152A - Compositions and methods for intestinal exo-miRNA expression profiling - Google Patents

Abstract

The present invention relates to compositions and methods for the analysis of microrna (miRNA) expression profiles in colorectal cancer plasma intestinal exosomes. In particular, the invention relates to a blood molecular marker diagnostic kit for diagnosing colorectal cancer, monitoring cancer treatment and/or treating colorectal cancer, the kit comprising polynucleic acid molecules, each nucleic acid molecule encoding a microrna sequence, wherein one or more nucleic acid molecules are differentially expressed in colorectal cancer plasma and healthy control plasma, 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 invention further relates to corresponding methods of using the nucleic acid expression signatures to determine colorectal cancer while preventing or treating such disorders. Finally, the present invention is directed to pharmaceutical compositions for the prevention and/or treatment of colorectal cancer.

Description

Compositions and methods for intestinal exo-miRNA expression profiling

Technical Field

The invention belongs to the field of biotechnology, and relates to a composition and a method for colorectal cancer plasma intestinal exosome microRNA (intestinal exosome microRNA, for short intestinal exo-miRNA expression profile analysis

Background

Colorectal cancer (colorectal cancer, CRC) is documented as one of the most common tumors worldwide. It was counted that in chinese new tumor, the CRC rank had risen to the second (12.2%), and in the new tumor death cases CRC rank was fifth (9.5%), up to 59.4% of patients had metastasized (lnadomi j. (2020). Gatstroenterology,158 (2): 289-290). CRC is curable if it can be diagnosed early in the progression of the lesion. However, in the early stages of the disease, most patients have no symptoms of the disease, so screening is necessary for early detection of CRC. Evidence suggests that early detection of CRC can significantly reduce its morbidity and mortality (Anwar, r. (2006) DIGESTIVE AND LIVER DISEASE, 34, 279-282).

Initially, CRC is characterized by the appearance of hyper-hyperplasia (atypical hyperplasia) in intestinal epithelial cells, with lesions first forming inflammatory adenomatous polyps in the colon or rectal intima, and then becoming abnormal neoplasm adenomas (i.e., benign tumors). In general, only a small subset of cells become malignant after adenomatosis (incidence about 60-70% at age 60), while more than 95% of cases of CRC have been demonstrated to be adenocarcinoma (Muto, T.et al. (1975) Cancer 36, 2251-2270;Fearon,E.R.and Vogelstein,B. (1990) Cell 61, 759-767).

Currently accepted methods of CRC screening include colonoscopy and fecal occult blood testing. Both tests then have their serious drawbacks. Colonoscopy, while effective, is a long-felt examination by many people due to its high cost, strong discomfort and potential side effects. On the other hand, the fecal occult blood test is a simple and inexpensive test, but the results are relatively inaccurate. Regardless, no specific molecular markers have been found so far that can be used to reliably diagnose CRC, particularly CRC that has been demonstrated to be adenocarcinoma, and/or CRC in the process of being converted from benign adenoma to malignant tumor.

Thus, the discovery of new biomarkers would be of great clinical importance, particularly if such markers could enable diagnosis early in tumor progression, allowing early treatment of cancer. Ideally, these markers should be capable of allowing the identification of cancer at a stage where the presence of malignant cells has not been detected by in situ techniques or microscopic analysis of biopsy or resected material.

Many diagnostic tests are also limited, which are typically based on analysis of only a single molecular marker, which may affect detection reliability and/or accuracy. In addition, individual markers often do not allow for detailed predictions regarding latency, tumor progression, etc. Thus, there is still a continuing need to identify other molecular markers and other assay formats that overcome these limitations.

One approach to this problem may be based on small regulatory RNA molecules, particularly microRNAs (miRNAs), which are a class of evolutionarily conserved endogenous expressed non-coding small RNAs, 20-25 nucleotides (nt) in size, that regulate the expression of target mRNAs. Since they were found about 10 years ago, mirnas were considered to have important functions .(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 cell development, differentiation, proliferation and apoptosis and, in addition, since they are stable in vitro and exist for a long time in vivo, they are more advantageous than mrnas as cancer markers (Lu, j.et al (2005) Nature 435, 834-838;Lim,L.P.et al (2005) Nature 433, 769-773).

Mirnas can direct different regulatory processes, depending on the degree of complementarity between the miRNA and its target. Target mRNAs highly complementary to miRNAs are specifically cleaved by the same mechanism as RNA interference (RNAi). The available existing data indicate that miRNAs play a role in cancer as protooncogenes or oncogenes, such as the overexpression of mir-17-92 in cancer plays a role in protooncogenes, promoting cancer progression, down-regulating tumor suppressor genes and/or genes controlling cell differentiation and apoptosis; it also down-regulates let-7a expression, whereas let-7a acts as a tumor suppressor gene by regulating protooncogenes and/or genes controlling cell differentiation and apoptosis (Zhang, b. (2007) Dev Biol 302,1-12). These demonstrate the role of mirnas in the progression of cancer. .

High-throughput miRNA quantification techniques such as miRNA chips, real-time RT-PCR-based TaqMan fluorescent quantitative detection of miRNA and the like provide a powerful tool for researching miRNA expression profiles in the whole cancer genome. There is growing evidence that many mirnas are deregulated in expression in cancer, including leukemia, lymphoma, glioblastoma, colon cancer, lung cancer, breast cancer, prostate cancer, thyroid cancer, liver cancer, ovarian cancer, and that they are differentially expressed in normal and cancerous tissues (Zhang, l. (2008) Adv Exp Med Biol 622, 69-78). Thus, miRNA profiles can be used to design features that recognize a variety of cancers, suggesting that they can help further establish molecular diagnosis, prognosis, and treatment. Abnormal expression of mirnas in cancer suggests that these mirnas may be useful for biomarkers and targets for molecular therapy.

Of the many possible sample types, blood is optimal for screening high risk populations, and it can be readily collected in a minimally invasive manner for early detection, early diagnosis, monitoring and effective treatment of cancer. It has been demonstrated that mirnas produced by tumour cells exist in a fairly stable form in human plasma or serum, which is protected from endogenous RNase degradation. The level of miRNA produced by tumor cells in plasma or serum is sufficient to be measured and used as a biomarker for cancer discovery. Furthermore, the levels of mirnas in plasma and serum are strongly correlated, indicating that clinical application of mirnas as biomarkers for cancer diagnosis, whether with plasma or serum samples, is suitable (Mitchell,P.S.et al.(2008)Proc Natl Acad Sci USA 105,10513-10518;Gilad,S.et al.(2008)PLoS ONE 3,e3148;Chen,X.et al.(2008)Cell Res 18,997－1006).

Several studies reported that the expression profile (Chen,X.(2008)Cell Res 18,997-1006;Ng EK.O.(2009)Gut 58,1375-1381;Huang Z.2009,Iht J Cancer,published on line). of plasma or serum mirnas in human CRC more than 100 circulating mirnas could be detected in the blood of healthy humans (Mitchell, p.s. (2008) Proc NATL ACAD SCI USA 105, 10513-10518), which is clearly different from the expression profile of colorectal cancer patients, who have several tumor specific mirnas. Old and colleagues demonstrated that 69 mirnas in serum of colorectal cancer patients were not available to healthy people (Chen, x. (2008) Cell Res 18, 997-1006). However, such large expression profiles are difficult to apply to clinical screening and diagnosis.

However, peripheral blood free mirnas are derived from various tissue cells: epithelial cells, fibroblasts, vascular endothelial cells, and a large number of leukocytes, especially in the case of tissue injury and inflammation. In addition, if the standardized operations of blood sampling and transportation are not strictly followed, the detection of peripheral blood free miRNA is interfered by cell rupture and released nucleic acid in the blood coagulation process, so that the accuracy, the reliability and the repeatability of clinical tests are difficult to ensure.

Exosomes, which are the latest research targets of "liquid biopsies", are small molecular membrane vesicles (40-100 nm in diameter) actively secreted from eukaryotic cells into the cell microenvironment, and are widely found in various body fluids such as blood and urine. The biogenesis of exosomes originates from the endosomal system, i.e. sustained invagination of the plasma membrane results in the formation of multivesicular bodies (multivesicular bodies, MVBs) which subsequently fuse MVBs with the plasma membrane, triggering exosome release. In this process, functional substances such as nucleic acids (mirnas), lipids, proteins, etc. are selectively encapsulated into the exosome cavity, and the bilayer membrane structure of the exosome can protect the encapsulated substances from extracellular degradation, increasing its stability. Research proves that miRNA in peripheral blood exosomes is more free and more stable than peripheral blood, so that miRNA in the exosomes can be used as a secretion molecule to influence the phenotype of receptor cells, can also be combined with surface receptors of specific cells through exosomes to enter target cells and regulate the target cells, and can also be used as a marker to play an important role in tumor diagnosis and treatment.

Studies have shown that miRNAs in exosomes are specifically expressed in CRC patients, such as miR-17, miR-18a, miR-19b, miR-92a, miR-375, miR-10b and miR-100. However, miRNA sources in blood exosomes are extensive, most of them are derived from blood cells, and organ or tissue specificity is difficult to achieve, affecting diagnostic specificity to some extent. Studies have shown that exosomes of different tissue cell sources carry markers of tissue specificity and disease specificity, and that some of them are detectable on the surface of exosomes. For example, GPC-1 related to pancreatic cancer, CD171 specific to nerve tissue, A33 specific to intestinal tissue, etc., are all exocrine surface protein markers having a certain tissue specificity.

However, currently detected exosome mirnas are all detected from total exosomes in peripheral blood, not tissue-specific exosomes. While most of the exosomes in the blood originate from blood cells, this will undoubtedly have a negligible effect on the detection of CRC markers. The rapid, simple and practical tissue-specific (i.e., intestinal-source-specific) in-vitro molecular markers that are clinically useful were mined and found to be capable of improving the specificity of CRC hematological screening and early diagnosis.

Thus, there remains a need to find diagnostic markers for miRNA of intestinal exosomes in the plasma or serum of a group of colorectal cancer patients. And combining a plurality of miRNA markers to establish a miRNA expression profile (fingerprint) based on blood samples, so that patients with colorectal cancer can be identified noninvasively, rapidly, accurately and economically. In addition, a corresponding method is continuously needed to screen out the high risk group of early colorectal cancer; early detection of recurrence of cancer; and/or monitoring cancer treatment.

Disclosure of Invention

It is an object of the present invention to provide novel methods for diagnosing colorectal cancer, monitoring cancer treatment and/or treating colorectal cancer by determining polynucleic acid molecules in blood intestinal tissue derived (a 33 positive) exosomes, each nucleic acid molecule encoding a microrna (miRNA) sequence, wherein one or more polynucleic acid molecules are differentially expressed in the plasma of a patient suffering from colorectal cancer, as compared to healthy controls and/or healthy humans, wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature, which signature is indicative of the occurrence of colorectal cancer, wherein the nucleic acid expression signature comprises tumor-related signature and plasma-specific signature.

Further, it is an object of the present invention to provide a corresponding method for identifying one or more nucleic acid expression characteristics in a blood sample exhibiting or having colorectal cancer. More specifically, it is an object of the present invention to provide a method to distinguish colorectal cancer from healthy controls and/or healthy persons.

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

In a first aspect, the invention relates to a diagnostic kit for identifying molecular markers in an intestinal (a 33 positive) exosome that shows or has colorectal cancer, the kit comprising polynucleic acid molecules, each encoding a microrna sequence, wherein one or more polynucleic acid molecules are differentially expressed in plasma of interest and in healthy controls, the differential expression profile being derived from a tumor-associated or plasma-specific profile, wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression profile that is indicative of colorectal cancer occurrence.

The nucleic acid expression profile as defined herein may comprise at least five nucleic acid molecules, preferably at least ten nucleic acid molecules, particularly preferably at least eighteen nucleic acid molecules.

Nucleic acid expression profiling includes at least one nucleic acid molecule encoding a microRNA sequence whose expression is up-regulated in one or more plasma of interest as compared to one or more healthy controls; and at least one nucleic acid molecule encoding a microRNA sequence whose expression is down-regulated in one or more plasmas of interest as compared to one or more healthy controls.

In embodiments, the nucleic acid expression signature comprises any one or more nucleic acid encoding plasma intestinal source (a 33 positive) exosome-specific signatures: nucleic acid molecules of hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-124-3p, hsa-miR-150-5p and hsa-miR-184, and an internal stability control: RNU6.

In embodiments, the expression of any one or more nucleic acid molecules encoding hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-124-3p and hsa-miR-184 is upregulated and the expression of any one or more nucleic acid molecules of miR-150-5p is downregulated in one or more plasma intestinal-source (A33-positive) exosomes of interest, as compared to one or more healthy controls, and RNU6 expression is unchanged.

In further preferred embodiments, the nucleic acid expression signature comprises any one or more of the nucleic acid encoding plasma intestinal source (A33 positive) exosome-specific signature ：miR-21-5p,hsa-miR-92a-3p,hsa-miR-122-5p,hsa-miR-124-3p,hsa-miR-146a-5p,miR-150-5p,hsa-miR-184,hsa-miR-196a-5p,miR-204-5p and miR-486-5p, and an internal stability control: RNU6.

In preferred embodiments, the expression of any one or more nucleic acid molecules encoding hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-124-3p, hsa-miR-184, hsa-miR-196a-5p, miR-204-5p and miR-486-5p is upregulated and the expression of any one or more nucleic acid molecules of hsa-miR-146a-5p, hsa-miR-122-5p and miR-150-5p is downregulated, and RNU6 expression is unchanged, in one or more healthy controls, as compared to one or more healthy controls.

In a second aspect, the invention relates to a method for identifying one or more plasma intestinal source (a 33 positive) exosomes of interest exhibiting or having colorectal cancer, the method comprising: (a) Determining the expression level of a polynucleic acid molecule in one or more plasma intestinal source (a 33 positive) exosomes of interest, each nucleic acid molecule encoding a microrna sequence; (b) Determining the expression level of the polynucleic acid molecules in one or more healthy control plasma intestinal source (a 33 positive) exosomes; and (c) identifying from the plurality of nucleic acid molecules one or more nucleic acid molecules that are differentially expressed in intestinal (a 33 positive) exosomes of the plasma of interest and control plasma by comparing the respective expression levels obtained in steps (a) and (b), wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature as defined herein that is indicative of the presence of colorectal cancer.

In a preferred embodiment of the invention, the method comprises: (a) Determining the expression level of a combination of nucleic acid molecules in one or more plasma intestinal source (a 33 positive) exosomes of interest, each nucleic acid molecule encoding a microrna sequence, and then calculating using a specific formula; (b) Determining the expression level of the combination of nucleic acid molecules in healthy control plasma intestinal source (a 33 positive) exosomes, and then calculating with a specific formula; and (c) identifying differences in the combination of nucleic acid molecules in the one or more plasma of interest and in the control plasma intestinal source (a 33 positive) exosomes by comparing the respective calculations obtained in steps (a) and (b), wherein the combination of one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature as defined herein, which nucleic acid expression signature is indicative of the presence of colorectal cancer.

In a more preferred embodiment of the invention, the method is further used to distinguish colorectal cancer from healthy individuals.

In a fourth aspect, the invention relates to a method for monitoring colorectal cancer treatment, the method comprising: (a) Identifying one or more nucleic acid expression characteristics in the plasma of interest using the methods defined herein; and (b) monitoring the expression level of one or more nucleic acid molecules in the blood that encode microRNA sequences that are within the combination of nucleic acid expression signatures, whereby post-treatment upregulation of plasma intestinal (A33 positive) exosome nucleic acid molecules with upregulation of pre-treatment expression is monitored following treatment expression downregulation and post-treatment upregulation of molecules with downregulation of pre-treatment expression.

In a fifth aspect, the invention relates to a method for preventing or treating colorectal cancer, the method comprising: (a) Identifying nucleic acid expression characteristics in plasma intestinal (a 33 positive) exosomes using the methods defined herein; and (b) modifying the expression level of one or more nucleic acid molecules in the blood that encode microRNA sequences that are within the combination of nucleic acid expression signatures such that expression of the nucleic acid molecules in the plasma intestinal source (A33 positive) exosomes is down-regulated and expression of the nucleic acid molecules in which expression is down-regulated is up-regulated.

In a sixth aspect, the present invention relates to a pharmaceutical composition for the prevention and/or treatment of colorectal cancer treatment in a blood sample, the composition comprising one or more nucleic acid molecules, each nucleic acid molecule encoding a sequence at least partially complementary to a microrna sequence encoded by a nucleic acid molecule whose expression is up-regulated in the plasma intestinal source (a 33 positive) exosomes of a patient suffering from colorectal cancer as defined herein, and/or a microrna sequence encoded by a corresponding nucleic acid molecule whose expression is down-regulated as defined herein.

Finally, in a seventh aspect, the present invention relates to the use of said pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of colorectal cancer.

Other embodiments of the present invention will become apparent from the detailed description that follows.

More specifically, the method comprises the steps of,

The present invention is based on the finding that colorectal cancer can be reliably identified based on the high sensitivity and accuracy of specific miRNA expression signatures in plasma intestinal (a 33 positive) exosomes, wherein said expression signatures as defined herein generally comprise up-and down-regulated human mirnas.

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

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are 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 in the following a group is defined to contain at least a certain number of embodiments, this is also to be understood as revealing a group preferably consisting of only these embodiments.

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

The term "about" in the present invention refers to an interval of accuracy within which a person skilled in the art understands that the technical effect of the object feature is still ensured. The term generally means + -10%, preferably + -5% of the deviation from the indicated value.

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

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

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

It is an object of the present invention to provide a novel method for diagnosing colorectal cancer, monitoring cancer treatment and/or treating colorectal cancer by determining in the blood polynucleic acid molecules, each encoding a microRNA (miRNA) sequence, wherein one or more polynucleic acid molecules are differentially expressed in the plasma intestinal source (A33 positive) exosomes of colorectal cancer as compared to healthy individuals, wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature which is indicative of the presence of colorectal cancer, including tumor-related signature and plasma-specific signature.

The term "colorectal" as used herein refers to the colon, rectum and/or appendix, i.e. the whole large intestine.

The term "cancer" (often referred to as "cancer") as used herein generally refers to any type of malignant neoplasm, i.e., any morphological and/or physiological change (based on genetic recombination) of a target cell that shows or has a propensity to develop a cancerous characteristic as compared to an unaffected (healthy) wild-type control cell. Examples of such changes may in particular relate to cell size and shape (becoming larger or smaller), cell proliferation (increasing cell number), cell differentiation (changing physiological state), apoptosis (programmed cell death) or cell survival. Thus, the term "colorectal cancer" refers to the cancerous growth of cells of the colon, rectum and appendix.

The most common colorectal (CRC) cell type is adenocarcinoma, accounting for 95% of cases. Other types of CRC include lymphomas and squamous cell carcinomas.

Colorectal cancer can be classified according to the Dukes system (Dukes, c.e. (1932) j.pathol.bacteriol.35, 323-325) by the following stages: dukes A-tumors are localized to the intestinal wall; dukes B-tumor invasion through the intestinal wall; dukes C-tumor invasion into lymph nodes: and Dukes' D-tumors have distant metastasis.

The term "plasma" as used herein is a yellow liquid component of blood on which red blood cells in whole blood are normally suspended. It is 55% by volume in whole blood, mostly water (90% by volume), and contains dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide (plasma is the main secretory product transport medium). The plasma is prepared by spin centrifugation of fresh blood until blood cells settle to the bottom of the tube, and then pouring or aspirating the plasma. The density of the plasma is approximately 1025kg/m ³, or 1.025kg/1. Current studies indicate that mirnas in plasma are stable. The term "plasma sample" refers to plasma obtained from a subject or healthy control.

The term "patient" as used herein refers to a person suspected of being at least colorectal cancer. As used herein, "plasma of interest" refers to plasma collected from a patient. The term "healthy individual" or "healthy control" generally refers to a healthy person without the cancerous phenotypic characteristics. As used herein, "control plasma" refers to plasma collected from a healthy individual. However, in other applications, such as when compared to other types of cancer, plasma collected from other types of cancer patients is often referred to as a "control".

Typically, the plasma sample used is obtained from a biological specimen, which is collected from a subject diagnosed with colorectal cancer. In addition, to obtain data for "comparison samples," other subjects of known disease states are also collected. Biological specimens include body tissues and fluids such as colorectal tissue, serum, blood cells, sputum, and urine. Alternatively, the biological sample may be obtained from an individual having an existing colorectal cancer trait or an individual suspected of having colorectal cancer. Moreover, if necessary, the obtained body tissue and liquid sample are purified and then used as biological samples. According to the present invention, a biological sample obtained from a subject determines the expression level of a nucleic acid marker of the present invention.

The samples to be tested in the present invention by in vitro methods should generally be collected in a clinically acceptable manner, in particular with respect to preserving nucleic acids (in particular RNA) or proteins. The sample to be analyzed is typically blood. In addition, lung tissue and other types of samples are also used. The samples may be combined, particularly after initial processing. However, uncombined samples may 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, 522-531). A "microrna" refers to the processing of an RNA molecule derived from a locus from a transcript to form a localized RNA precursor miRNA structure. Mature mirnas are typically 20, 21, 22, 23, 24 or 25 nucleotides in length, and other numbers of nucleotides may be present, such as 18, 19, 26 or 27 nucleotides.

MiRNA coding sequences have the potential to pair with flanking genomic sequences, allowing mature mirnas to be placed within incompletely paired RNA duplex (also referred to herein as stem-loop or hairpin structures or pre-mirnas) that serve as intermediates for miRNA processing from longer precursor transcripts. This processing generally occurs by the sequential action of two specific endonucleases known as Drosha and Dicer, respectively. Drosha produces a miRNA precursor (also referred to herein as a "pre-miRNA") from a primary transcript (also referred to herein as a "pri-miRNA") that is typically folded into a hairpin or stem-loop structure. From this miRNA precursor, the miRNA duplex is cleaved by Dicer, which comprises the mature miRNA in one arm of the hairpin or stem-loop structure and a similarly sized fragment (commonly referred to as miRNA x) in the other arm. mirnas are then targeted to their target mrnas to perform their function, whereas mirnas are degraded. In addition, mirnas are generally derived from genomic fragments that differ from the predicted protein coding region.

The term "miRNA precursor" (or "precursor miRNA" or "pre-miRNA") as used herein refers to the portion of the miRNA primary transcript from which the mature miRNA is processed. In general, pre-mirnas fold into stable hairpin (i.e., duplex) or stem-loop structures. Hairpin structures are typically 50-80 nucleotides in length, preferably 60-70 nucleotides (counting miRNA residues, those paired with miRNA 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 the corresponding precursor mirnas and primary miRNA transcripts as described above. In addition, the 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. The nucleic acid molecules of the invention encoding microrna sequences typically encode a single miRNA sequence (i.e., an individual miRNA). However, it is also possible that such nucleic acid molecules encode two or more miRNA sequences (i.e. two or more mirnas), e.g. one transcription unit comprises two or more miRNA sequences under the control of common regulatory sequences such as promoters or transcription terminators.

The term "nucleic acid molecule encoding a microRNA sequence" as used herein is also understood to include "sense nucleic acid molecules" (i.e., molecules whose nucleic acid sequence (5 '. Fwdarw.3') matches or corresponds to the encoded miRNA (5 '. Fwdarw.3') sequence) and "antisense nucleic acid molecules" (i.e., molecules whose nucleic acid sequence is complementary to the encoded miRNA (5 '. Fwdarw.3') sequence or in other words matches the reverse complement (3 '. Fwdarw.5') of the encoded miRNA sequence). 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 (i.e. "sense" and "antisense" molecules) may be fully complementary, i.e. they do not contain any base mismatches and/or additional or missing nucleotides. Either 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 consecutive nucleotides exhibiting complete complementarity to a sequence comprised in a corresponding "sense" nucleic acid molecule.

Thus, the polynucleic acid molecules encoding miRNA sequences comprised in the diagnostic kit of the invention may comprise one or more "sense nucleic acid molecules" and/or one or more "antisense nucleic acid molecules". Sometimes, a diagnostic kit comprises one or more "sense nucleic acid molecules" (i.e. the miRNA sequences themselves) which are considered to constitute the totality or at least a subset of differentially expressed mirnas (i.e. molecular markers) which are indicative of the presence or propensity for developing a particular disorder, here colorectal cancer. On the other hand, when the diagnostic kit comprises one or more "antisense nucleic acid molecules" (i.e., sequences complementary to miRNA sequences), the molecules may comprise probe molecules (for performing hybridization assays) and/or oligonucleotide primers (e.g., for reverse transcription or PCR applications) suitable for detecting and/or quantifying one or more specific (complementary) miRNA sequences in a given sample.

The polynucleic acid molecules identified 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 encoding a miRNA sequence.

The term "intestinal exosomes" as used herein is also understood to include "intestinal tissue-derived exosomes", meaning Extracellular Vesicles (EV) actively released by intestinal epithelial cells, having a diameter of 40-160nm (average 100 nm). The biogenesis of exosomes originates from the endosomal system, i.e. sustained invagination of the plasma membrane leads to the formation of MVB, which then fuses with the plasma membrane, triggering exosome release. In addition, exosomes contain a number of bioactive ingredients including DNA, RNA, lipids, proteins, amino acids and metabolites. Various RNAs are also found in exosomes, including mirnas, mrnas and other ncrnas. Among them, mirnas are of great interest due to their high conservation among species and wide regulation in gene expression. As short fragments (20-24 nt) of non-coding RNA, mirnas can affect translation and stability of mRNA, thereby regulating gene expression in multicellular organisms at the post-transcriptional level.

The term "A33 positive" as used herein refers to the expression of the A33 protein on the surface of an exosome. A33, also known as GPA33, is a transmembrane immunoglobulin which is expressed in both normal intestinal epithelium and more than 95% of carcinoma epithelial cells of the large intestine and has increased expression in large intestine (Suresh M.mol Cell Proteomics,2010,9 (2): 197-208.). We therefore selected the A33 extracellular segment (aa 150-200) as the target for intestinal exosome capture.

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

Within the scope of the present invention, a nucleic acid molecule is considered differentially expressed if the respective expression levels of the nucleic acid molecule in a disease plasma sample and a control sample generally differ by at least 5% or by at least 10%, preferably by at least 20% or by at least 25%, most preferably by at least 30% or by at least 50%. Thus, the latter value corresponds to an up-regulation of the expression level of a given nucleic acid molecule in a disease plasma sample by at least 1.3-fold or at least 1.5-fold compared to a control plasma sample, respectively, or vice versa in a disease plasma sample by at least 0.7-fold or at least 0.5-fold.

The term "expression level" as used herein refers to the extent to which a particular miRNA sequence is transcribed from its genomic locus, i.e., the concentration of miRNA in the plasma sample to be analyzed.

As described above, the term "control plasma" generally refers to plasma collected from (healthy) individuals without a phenotypic characteristic of colorectal cancer. However, in certain applications, plasma collected from other types of cancer patients is generally referred to as "control plasma" as compared to other types of cancer.

The determination of expression levels can generally follow established standard procedures (Sambrook,J.et a1.(1989)Molecular Cloning：A Laboratory Mamual.2nd Ed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY;Ausubel,F.M.et al.(2001)Current Protocols in Molecular Biology.Wiley&Sons,Hoboken,NJ). well known in the art, such as Northern blot analysis with miRNA-specific probes, or at the DNA level after reverse transcription (and cloning) of RNA populations, such as by quantitative PCR or real-time PCR techniques. The term "determining" as used herein includes the analysis of any nucleic acid molecule encoding a microRNA sequence as described above. However, due to the short half-life of pri-miRNA and pre-mRNA, only the concentration of mature miRNA is typically measured.

In specific embodiments, standard values of expression levels obtained in several independent measurements (e.g., two, three, five, or ten measurements) of a given sample and/or in several measurements of multiple samples or control samples are used for analysis. The standard values may be obtained by any method known in the art. For example, a range of the average value ±2sd (standard deviation) or the average value ±3sd may be used as the standard value.

The difference between the obtained expression levels in the disease plasma and the control plasma may be normalized to the expression level of a further control nucleic acid, e.g. housekeeping gene, which is known to be not different depending on the disease state of the individual from which the sample was collected. Typical housekeeping genes include beta-actin, glyceraldehyde-3-phosphate deoxygenase, ribosomal protein P1, and the like. In a preferred embodiment, the control nucleic acid is another miRNA known to be stably expressed in various non-cancerous and precancerous (pre) conditions in the individual from which the sample was collected.

However, instead of determining the expression level of a plasma sample in any experiment, one or more cut-off values for a specific disease phenotype (i.e. disease state) may also be defined based on experimental evidence and/or prior art data. In this case, the respective expression levels in the plasma samples can be determined using normalized stably expressed control mirnas. If the calculated "normalized" expression level is higher than the corresponding defined cutoff value, this finding is indicative of an up-regulation of gene expression. Conversely, if the calculated "normalized" expression level is below the correspondingly defined cutoff value, then this finding is indicative of down-regulation of gene expression.

In the present invention, one or more differentially expressed nucleic acid molecules identified by a plasma sample together represent a nucleic acid expression signature that is indicative of colorectal cancer. The term "expression signature" as used herein refers to a group of nucleic acid molecules (e.g., mirnas) wherein the expression level of each nucleic acid molecule varies between the plasma of colorectal cancer patients and the plasma intestinal (a 33 positive) exosomes of healthy controls. Nucleic acid expression signature is also referred to herein as a set of markers and represents the minimum number of (different) nucleic acid molecules, each encoding a miRNA sequence that identifies the phenotypic status of an individual.

In a first aspect, the invention relates to a diagnostic kit for molecular markers in blood, the kit comprising polynucleic acid molecules, each nucleic acid molecule encoding a microrna sequence, wherein one or more of the polynucleic acid molecules in the intestinal source (a 33 positive) exosomes of the plasma of interest are differentially expressed compared to healthy controls, wherein the characteristic of the differential expression is derived from a tumor-associated or plasma-specific characteristic, wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression characteristic that is indicative of the presence of colorectal cancer.

The nucleic acid expression profile described herein may comprise at least seven nucleic acid molecules, preferably at least eleven nucleic acid molecules, and particularly preferably at least nineteen nucleic acid molecules.

In a preferred embodiment, the nucleic acid expression profile comprises at least one nucleic acid molecule encoding a microRNA sequence, which microRNA expression is up-regulated in one or more plasma intestinal source (A33 positive) exosomes of interest as compared to one or more healthy controls, and at least one nucleic acid molecule encoding a microRNA sequence, which microRNA expression is down-regulated in one or more plasma intestinal source (A33 positive) exosomes of interest as compared to one or more healthy controls.

Colorectal cancer tissue cells described herein are cancerous large intestine cells collected from an isolated subject diagnosed with colorectal cancer. The non-cancerous tissue cells are typically derived from a (healthy) wild-type cell that does not have the cancerous phenotypic characteristic.

Typically, the nucleic acid molecule that is included in the nucleic acid expression profile is a human sequence (hereinafter "hsa" (human).

In preferred embodiments, the nucleic acid expression signature comprises any one or more nucleic acid molecules encoding a tumor-associated signature ：hsa-miR－146a-5p(SEQ ID NO：1),hsa-miR-486-5p(SEQ ID NO：2),hsa－miR-122-5p(SEQ ID NO：3),hsa-miR－184(SEQ ID NO：4,hsa-miR-196a-5p(SEQ ID NO：5,hsa-miR-150-5p(SEQ ID NO：6,hsa-miR-21－5p(SEQ ID NO：7,hsa-miR-26a-5p(SEQ ID NO：8,hsa-miR-124-3p(SEQ ID NO：9,hsa-miR-204-5p(SEQ ID NO：10.

To homogenize plasma expression levels, nucleic acid molecules encoding microRNA sequences comprised in the nucleic acid expression profile, RNU6 (SEQ ID NO: 11) should be preferentially used, which are stably expressed in colorectal cancer plasma intestinal (A33 positive) exosomes.

The nucleic acid sequences of the above mirnas are listed in table 1.

TABLE 1

All miRNA sequences disclosed herein have been stored in the milbase database (http:// microma. Sanger. Ac. Uk/; see also Griffiths-Jones S.et al (2008) nucleic acids Res.36, D154-D158).

In a more preferred embodiment, the expression of any one or more nucleic acid molecules encoding hsa-miR-146a-5p, hsa-miR-122-5p and miR-150-5p is upregulated in one or more blood plasma of interest, relative to one or more healthy controls, and the expression of hsa-miR-124-3p, hsa-miR-184, hsa-miR-196a-5p, miR-204-5p and miR-486-5p is downregulated; RNU6 expression was unchanged.

As used herein, the terms "any one or more polynucleic acid molecules" and "one or more of the nucleic acid molecules" may refer to any subset of the polynucleic acid molecules, e.g., any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten, etc., nucleic acid molecules, each nucleic acid molecule encoding a microrna sequence comprised within the nucleic acid expression signature.

The term "nucleic acid combination" as used herein refers to the use of at least two levels of nucleic acid expression as a whole. The results may preferably be calculated as a whole with the relevant changes or by formulas.

In a second aspect, the invention relates to a method for identifying one or more plasma of interest that are indicative of or have colorectal cancer, the method comprising: (a) Determining the expression level of a polynucleic acid molecule in one or more plasma intestinal source of interest (a 33 positive) exosomes, each nucleic acid molecule encoding a microrna sequence; (b) Determining the expression level of the polynucleic acid molecules in one or more healthy control plasma; and (c) identifying from the plurality of nucleic acid molecules one or more nucleic acid molecules that are differentially expressed in intestinal (a 33 positive) exosomes of the plasma of interest and a control plasma by comparing the respective expression levels obtained in steps (a) and (b), wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature as specified herein that is indicative of the presence of colorectal cancer.

In a preferred embodiment of the invention, the method comprises: (a) Determining the expression level of a combination of nucleic acid molecules in one or more plasma intestinal source of interest (a 33 positive) exosomes, each nucleic acid molecule encoding a microrna sequence, and then calculating using a specific formula; (b) Determining the expression level of the combination of nucleic acid molecules in healthy control plasma intestinal source (a 33 positive) exosomes, and then calculating with a specific formula; and (c) identifying differences in the combination of nucleic acid molecules in one or more intestinal sources of plasma of interest and control plasma (a 33 positive) exosomes by comparing the respective calculations obtained in steps (a) and (b), wherein the combination of one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature as defined herein, which nucleic acid expression signature is indicative of the presence of colorectal cancer.

In a third aspect, the invention relates to a method for monitoring colorectal cancer treatment, the method comprising: (a) Identifying nucleic acid expression characteristics in one or more plasma intestinal source of interest (a 33 positive) exosomes using the methods defined herein; and (b) monitoring the expression level of one or more nucleic acid molecules in the blood that encode microRNA sequences that are within the combination of nucleic acid expression signatures, whereby the plasma intestinal source (A33 positive) exosome nucleic acid molecules that are up-regulated in pre-treatment expression are up-regulated after treatment and post-treatment with molecules that are down-regulated in pre-treatment expression.

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

In a fourth aspect, the present invention relates to a method for preventing or treating colorectal cancer, the method comprising: (a) Identifying nucleic acid expression characteristics in a plasma intestinal source (a 33 positive) exosome using the methods defined herein; and (b) modifying the expression level of one or more nucleic acid molecules in the blood that encode microRNA sequences that are within the combination of nucleic acid expression signatures such that expression of the nucleic acid molecules in the plasma intestinal source (A33 positive) exosomes is down-regulated and expression of the nucleic acid molecules in which expression is down-regulated is up-regulated.

In the present invention, the expression of one or more nucleic acid molecules encoding microRNA sequences comprised in the nucleic acid expression profile is modified in such a way that the expression of nucleic acid molecules whose expression is up-regulated in the plasma intestinal source (A33 positive) exosomes is down-regulated and the expression of nucleic acid molecules whose expression is down-regulated in the plasma is up-regulated. In other words, modification of expression of a particular nucleic acid molecule encoding a miRNA sequence to modulate the molecule in the plasma of a cancer patient occurs in a reverse circulation pattern to scramble the "overactivity" of the up-regulated molecule and/or to restore the "defective activity" of the down-regulated molecule.

In a preferred embodiment of the method of the invention, down-regulating 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 "introduced into the blood" as used herein refers to any manipulation that causes one or more nucleic acid molecules to migrate into the blood. Examples of such techniques include injection, digestive absorption, or any other technique that may be involved.

The term "complementary sequence" as used herein refers to a nucleic acid molecule which is understood to be "complementary" to the introduction of the nucleic acid molecule (also referred to herein as "antisense nucleic acid molecule") into the blood, which is capable of forming base pairs, preferably Watson-Crick base pairs, with an up-regulated endogenous "sense" nucleic acid molecule.

The two nucleic acid molecules (i.e., the "sense" and "antisense" molecules) may be fully complementary, i.e., they do not contain any base mismatches and/or additions or deletions of nucleotides. In other embodiments, the two molecules contain one or more base mismatches or the total number of nucleotides is different (due to additions or deletions). In a further embodiment, a "complementary" nucleic acid molecule comprises a stretch of at least ten consecutive nucleotides that is fully complementary to the sequence comprised in the up-regulated "sense" nucleic acid molecule.

A "complementary" nucleic acid molecule (i.e., a nucleic acid sequence encoding a nucleic acid molecule that is complementary to a nucleic acid sequence encoding a down-regulated microRNA sequence) can be a naturally occurring DNA-or RNA molecule or a synthetic nucleic acid molecule that includes one or more modified nucleotides of the same type or of one or more different types in its sequence.

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 to 2' -O-methyl groups or 2' -O-methoxy groups (also referred to as 2' -O-methylation), which prevent nuclease degradation in the culture medium and importantly also prevent core separation of RNA-induced silencing complex nucleases, resulting in irreversible inhibition of mirnas. Another possible modification, which is functionally equivalent to 2' -O-methylation, comprises a Locked Nucleic Acid (LNA), representing a nucleic acid analog containing one or more LNA nucleotide monomers having locked bicyclic furanose units in an RNA mimic sugar conformation (Orom, U.A. et al. (2006) Gene 372, 137-141).

Another class of miRNA expression silencing genes has recently been developed. These chemically engineered oligonucleotides, known as "antagomers", 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 are produced as RNAs from transgenes that can be expressed in cells. These competitive inhibitors, known as "microRNA sponges", are transcripts expressed from strong promoters, containing multiple tandem binding sites for the micrornas of interest (Ebert, m.s. et al (2007) nat. Methods 4, 721-726).

In a particularly preferred embodiment of the invention, the one or more nucleic acid molecules encoding microRNAs whose expression is down-regulated are selected from the group consisting of hsa-miR-146a-5p, hsa-miR-122-5p and miR-150-5p, and for which expression characteristics are likely to be indicative of colorectal cancer, as described above.

In a further preferred embodiment of the invention, up-regulating the expression of a nucleic acid molecule comprises introducing a nucleic acid molecule encoding a microRNA sequence encoded by the up-regulated nucleic acid molecule into the blood. In other words, upregulation of expression of a nucleic acid molecule encoding a miRNA sequence is achieved by introducing another copy of the miRNA sequence (i.e. an additional "sense" nucleic acid molecule) into the blood. The "sense" nucleic acid molecule introduced into the blood may comprise the same modifications as the "antisense" nucleic acid molecule described above.

In a particularly preferred embodiment, the one or more nucleic acid molecules encoding microRNAs whose expression is up-regulated are selected from the group consisting of hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-124-3p, hsa-miR-184, hsa-miR-196a-5p, miR-204-5p and miR486-5p, and expression features that are likely to be indicative of colorectal cancer, as described above.

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

To elucidate any potential association of identified mirnas in cancerous or pre-cancerous samples, a preliminary functional analysis can be performed regarding 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,CM.(2007)supra;Blenkiron,C.and Miska,E.A.(2007)supra),, it is speculated that the mRNA target site for such mirnas includes tumor suppressor genes and oncogenes.

A nucleic acid molecule is said to be "capable of expressing a nucleic acid molecule" or "causing expression of a nucleotide sequence" if the nucleic acid molecule comprises sequence elements that contain information about transcriptional and/or translational regulation, and such a sequence is "operably linked" to a nucleotide sequence encoding a polypeptide. An operable linkage is one in which the regulatory sequence elements are linked to the expressed sequence (and/or to each other) in a manner that enables expression of the gene.

The exact nature of the regulatory regions necessary for gene expression may vary among species, but typically these regions each comprise a promoter, which in prokaryotes contains two promoters, a DNA element that directs transcription initiation and a DNA element that signals translation initiation when transcribed into RNA. Such promoter regions typically include 5 'non-coding regions involved in transcription and translation initiation, such as the-35/-10 box and Shine-Dalgamo element in prokaryotes or the TATA box, CAAT sequence, and 5' -capping element in eukaryotic cells. These regions may also include enhancer or repressor elements and translational signals and leader sequences to target the native polypeptide to a particular compartment of the host cell. In addition, the 3' non-coding sequence may contain regulatory elements involved in transcription termination, polyadenylation, and the like. However, if these termination sequences are not satisfactory for function in a particular host cell, they may be replaced with signals that function in that cell.

Furthermore, the expression of a nucleic acid molecule as defined herein may also be affected (as described above) by, for example, the presence of modified nucleotides. 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 miRNA-target duplex structures critical for silencing activity (Naguibneva, 1.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, optionally also transcription termination sequences. The promoter may allow constitutive or inducible gene expression. Suitable promoters include the E.coli (E.coli) lacUV5 and tet (tetracycline response) promoters, the T7 promoter, the SV40 promoter or the CMV promoter.

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

In a fifth aspect, the present invention relates to a pharmaceutical composition for the prevention and/or treatment of colorectal cancer treatment in a blood sample, the composition comprising one or more nucleic acid molecules, each nucleic acid molecule encoding a sequence at least partially complementary to a microrna sequence encoded by a nucleic acid molecule whose expression is up-regulated in the plasma intestinal source (a 33 positive) exosomes of a colorectal cancer patient as defined herein, and/or a microrna sequence encoded by a corresponding nucleic acid molecule whose expression is down-regulated as defined herein.

Finally, in a sixth aspect, the present invention relates to the use of said pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of cancer.

Within the scope of the present invention, suitable pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), peritoneal and parenteral (including intramuscular, subcutaneous or intravenous) administration, or those administered by inhalation or insufflation. The administration may be local or systemic. Preferably by oral or intravenous route. The formulations may be packaged as individual dosage units.

Pharmaceutical compositions of the invention include any pharmaceutical dosage form identified in the art, such as capsules, microcapsules, cachets, pills, tablets, powders, pellets (pellets), multiparticulate formulations (e.g., 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 emulsions, lotions and balsams.

The above-described ("sense" and "antisense") nucleic acid molecules can be formulated into pharmaceutical compositions using pharmaceutically acceptable ingredients and established methods of preparation (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 Mamufacturing Formulations,CRC Press,Boca Raton,FL).

For the preparation of the pharmaceutical composition, pharmaceutically inert inorganic or organic excipients (i.e. 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, lipo-acids, waxes, solid or liquid polyols, natural oils or hardened oils. Suitable excipients for the production of solutions, suspensions, emulsions, aerosol mixtures or powders which are reconstituted into solutions or aerosol mixtures prior to use include water, alcohols, glycerol, polyols and suitable mixtures thereof, and vegetable oils.

The pharmaceutical compositions may also contain additives such as fillers, binders, moisturizers, glidants, stabilizers, preservatives, emulsifiers, and additional solvents or solubilizers or substances for achieving a depot effect. The latter is understood to mean that the nucleic acid molecules can be incorporated into slow-release or sustained-release or targeted delivery systems such as liposomes, nanoparticles and microcapsules.

In order to target most tissues in the body, a clinically viable non-invasive strategy is required to target such pharmaceutical compositions as defined herein to cells. In the past, some methods have achieved significant therapeutic benefit by intravenous injection of reasonable doses of siRNA into mice and primates without significant limiting toxicity.

One method involves covalently coupling the passenger strand of miRNA (miRNA strand) with cholesterol or its derivatives/conjugates to promote absorption by ubiquitously expressed cell surface LDL receptors (Soutschek, j.et al (2004) Nature 432, 173-178). Or unconjugated PBS-formulated locked nucleic acid modified oligonucleotides (LNA-anti-r) can be delivered systemically (Elmen, j.et al (2008) Nature 452, 896-899). Another method of delivering mirnas involves encapsulation of mirnas into specific liposomes using polyethylene glycol to reduce uptake by cleared cells and enhance circulation time. These specific nucleic acid particles (stabilized nucleic acid-lipid particles or SNALP) efficiently deliver mirnas to the liver (and not to other organs (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 conjugated addition of alkyl acrylates or alkyl-acrylamides to primary or secondary amines) have been described as delivery agents for RNAi therapeutics (Akinc, a.et a1. (2008) nat. Biotechnol.26, 561-569).

A further cell-specific targeting strategy involves mixing miRNA with a fusion protein consisting of a targeting antibody fragment linked to protamine, an alkaline protein that nucleates DNA in the pins and binds miRNA by charge (Song, e.et al (2005) nat. Biotechnol.23, 709-717). Various modifications and changes to the basic conveying method described above have been recently developed. Such techniques are known in the art and are reviewed in, for example, 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 invention is further described by the accompanying drawings and examples which are provided for the purpose of illustrating specific embodiments of the invention and are not to be construed as limiting the scope of the invention in any way.

Drawings

FIG. 1 depicts a flow chart schematically illustrating the basic method steps for determining the expression profile of the present invention for identifying one or more plasma intestinal source (A33 positive) exosome microRNAs of interest that are indicative or have colorectal cancer.

FIG. 2 illustrates ROC curve analysis, using quantitative RT-PCR to detect two tumor-associated miRNAs (hsa-miR-92 a-3p and miR-150-5 p) of colorectal cancer target plasma and healthy control plasma intestinal source (A33 positive) exosomes, and shows that these plasma intestinal source (A33 positive) exosomes miRNAs are highly sensitive and specific as diagnostic markers.

Detailed Description

Example 1: plasma sample collection and preparation

The subjects collected fasting venous blood 10ml, edta was anticoagulated, and plasma was centrifuged twice at 800g x 10min and 16000g x 10min in 2 hours. Taking 650 μL of plasma, centrifuging for 15min at 3000g, removing precipitate and lipid layer, taking 600 μL of supernatant in a new EP tube, centrifuging for 10min at 16000g to avoid gel blockage by large particulate matters, and finally storing the plasma sample at-80 ℃ for later use.

The total plasma exosomes were enriched by gel exclusion chromatography (BE-SEC column, jiangsu is a company of Probiotics technology Co., ltd., 50620001) according to the manufacturer's instructions, and the intestinal exosomes were purified by adsorption using Anti-A33 protein extracellular fragment antibody ab 197370-labeled magnetic beads (Jiangsu is a company of Probiotics technology Co., ltd., 50630004). Total RNA was extracted from plasma using miRNeasy Micro Kit isolation kit (QIAGEN, germany). Quantification was performed with a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, waltham, MA). Quality control was performed by a 2100 bioanalyzer using an RNA 6000Pico LabChip kit (Agilent Technologies, SANTA CLARA, CA).

Table 2 shows the baseline characteristics of the blood samples used in the present invention. All patient samples were obtained surgically. Patient data (age, sex, image data, treatment method, other medical conditions, family history, etc.) are obtained from the hospital database. The tumor histopathies are classified by three pathologists according to the world health organization tumor classification system, respectively.

TABLE 2

Reference features of blood specimens

Whole genome miRNA analysis	Quantity of
		Healthy individuals	5
Colorectal cancer	6
		Quantitative RT-PCR analysis
Healthy individuals	5
		Colorectal cancer	6
Total number of	22

Example 2: analysis of miRNA expression profiles in plasma samples

Sequencing analysis was performed on (differentially) expressed mirnas in 11 of the plasma samples using Illumina NovaSeq6000 second generation high throughput sequencing according to manufacturer's instructions. For each sample, no less than 20M of raw reads were generated, and the specific gravity of base mass greater than 20 (Q20) was greater than 98%. A total of 312 known miRNAs were detected by sequencing.

With P-value < = 0.05, fold-change (FC) > = 2, 18 differentially expressed plasma intestinal exosomes mirnas were screened (table 3). If these two criteria are met, the miRNAs are considered to be differentially expressed in colorectal cancer patients and healthy individuals, respectively.

TABLE 3 Table 3

Sequencing results show that the expression difference of plasma intestinal source exosome miRNA in colorectal cancer patients and healthy individuals is compared

/>

The CRC and healthy control miRNA differential expression assay data are summarized in table 4. Table 4 lists the significant differences in miRNA expression in CRC patient tissues and plasma compared to control tissues and healthy control plasma, respectively. "t" represents colorectal cancer tissue, "n" represents matched normal tissue, and "p" represents colorectal cancer patient, "h" represents healthy control. Particularly preferred miRNAs (SEQ ID NOS: 1-10 are set forth in Table 4: SEQ ID NOS 21to SEQ ID NO:24in Table 5,respectively) are indicated in bold.

TABLE 4 Table 4

Tumor-related miRNA (micro ribonucleic acid) characteristics in colorectal cancer plasma intestinal exosomes

/>

Note：infmeans‘∞’.

Example 3: verification of sequencing data

To verify (and/or quantify) the miRNA expression data obtained by sequencing, the verification was performed using the TB GREEN QPCR assay (Takara, japan) according to the manufacturer's instructions using established quantitative RT-PCR. Briefly, reverse Transcription (RT) was performed using the Mir-X MIRNA FIRST-STRAND SYNTHESIS KIT kit according to instruction of the specification. 200ng of total RNA was reverse transcribed in a 6.25ul RT solution mixture containing 2X mRQ Buffer, mRQ Enzyme, and the RT solution was then subjected to thermal cycling on a PCR instrument (THERMAL CYCLER ALPHA ENGINE, bio-rad) at 37℃for 60min and 85℃for 5 min. Quantitative PCR was performed using TB GREEN QPCR MASTER Mix kit according to instructions of the instructions. 2ul RT product was amplified in 2X TB Green Advantage qPCR Premix、PCR Forward Primer(10μM)、PCR Reverse Primer(10μM)、50X ROX Reference Dye LSR mixtures and each reaction was repeated twice. Real-time PCR was performed on a StepOnePlus System real-time fluorescent quantitative PCR apparatus from ThermoFisher Scientific, with the procedure of initial heating at 96℃for 30s, followed by 40 cycles of 95℃for 5s and 60℃for 30s. mirnas were relatively quantified by the 2 ^*ΔΔ^Ct method. Each miRNA Ct value was normalized by an internal stable control U6.

The results obtained demonstrate a global highly specific regulation of miRNA expression in colorectal cancer. Thus, the corresponding subsets of mirnas described herein represent unique miRNA expression signatures for expression profiling of colorectal cancer, which not only allows identification of the cancerous state itself, but also allows differentiation from hepatocellular and lung cancers.

The invention provides a unique molecular marker for monitoring, finding and diagnosing colorectal cancer through blood for determining miRNA expression characteristics. In addition, the expression profile can be used to monitor therapeutic response of colorectal cancer patients and guide therapeutic decisions. In addition, the expression characteristics can also be used for developing medicines for resisting colorectal cancer.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be construed broadly and without limitation. In addition, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by embodiments and optional features, modification and variation of the invention may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

Claims (10)

1. Use of a miRNA as a colorectal cancer detection or treatment target, characterized in that the miRNA is selected from one or more of miR-21-5p,hsa-miR-92a-3p,hsa-miR-122-5p,hsa-miR-124-3p,hsa-miR-146a-5p,miR-150-5p,hsa-miR-184,hsa-miR-196a-5p,hsa-miR-204-5p and hsa-miR-486-5 p.
2. 2. Use of a reagent that measures the expression level of a miRNA, said miRNA being one or more of miR-21-5p,hsa-miR-92a-3p,hsa-miR-122-5p,hsa-miR-124-3p,hsa-miR-146a-5p,miR-150-5p,hsa-miR-184,hsa-miR-196a-5p,hsa-miR-204-5p,hsa-miR-486-5p, in the preparation of a colorectal cancer diagnostic reagent that diagnoses whether a subject has or is at risk of colorectal cancer by measuring the expression level of said miRNA in a sample, wherein an up-regulation of hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-124-3p, hsa-miR-184, hsa-miR-196a-5p, hsa-miR-204-5p and/or hsa-miR-486-5p expression, or a-miR-146a-5p, hsa-miR-122-5p and/or hsa-miR-150-5p expression, compared to the corresponding expression level of a miRNA in a control sample, is indicative of a subject having or being at risk of developing colorectal cancer.
3. 3. A colorectal cancer detection kit, which is characterized in that the kit contains a reagent capable of detecting the expression level of miRNA, wherein the miRNA is miR-21-5p,hsa-miR-92a-3p,hsa-miR-122-5p,hsa-miR-124-3p,hsa-miR-146a-5p,miR-150-5p,hsa-miR-184,hsa-miR-196a-5p,hsa-miR-204-5p and/or hsa-miR-486-5p.
4. 4. The kit of claim 3, wherein the agent that detects the level of expression of a miRNA is a polynucleic acid molecule, each polynucleic acid molecule encoding or being partially or fully complementary to a corresponding miRNA sequence, said miRNA being miR-21-5p,hsa-miR-92a-3p,hsa-miR-122-5p,hsa-miR-124-3p,hsa-miR-146a-5p,miR-150-5p,hsa-miR-184,hsa-miR-196a-5p,hsa-miR-204-5p and/or hsa-miR-486-5p.
5. 5. The kit of claim 4, further comprising an internal stability control RNU6.
6. 6. The kit of claim 3, for detecting the level of expression of a miRNA in an exosome of plasma intestinal origin (A33 positive), wherein the miRNA is miR-21-5p, hsa-miR-92a-3p, hsa-miR-122-5p, hsa-miR-124-3p, hsa-miR-146a-5p, miR-150-5p,

   Hsa-miR-184, hsa-miR-196a-5p, hsa-miR-204-5p and/or hsa-miR-486-5p.
7. 7. Use of an agent for measuring the expression level of miRNA for monitoring the progress of colorectal cancer treatment, said agent comprising

   The miRNA is miR-21-5p, hsa-miR-92a-3p, hsa-miR-122-5p and hsa-miR-124-

   3p，hsa-miR-146a-5p，miR-150-5p，hsa-miR-184，hsa-miR-196a-5p，hsa-

   MiR-204-5p and/or hsa-miR-486-5p, and monitoring hsa-486-5 p in blood after treatment by using measuring reagent

   miR-21-5p、hsa-miR-92a-3p、hsa-miR-124-3p、hsa-miR-184、hsa-miR-

   Down-regulation of 196a-5p, hsa-miR-204-5p and/or hsa-miR-486-5p expression levels, or hsa-fluviograph

   The expression level of miR-146a-5p, hsa-miR-122-5p and/or hsa-miR-150-5p is up-regulated.
8. 8. Use of one or more polynucleic acid molecules that promote down-regulation of hsa-miR-21-5p, hsa-miR-92a-3p, hsa-miR-124-3p, hsa-miR-184, hsa-miR-196a-5p, hsa-miR-204-5p and/or hsa-miR-486-5p expression, or up-regulation of hsa-miR-146a-5p, hsa-miR-122-5p and/or hsa-miR-150-5p expression, in the preparation of a medicament for treating or preventing colorectal cancer.
9. 9. The use according to claim 8, wherein the one or more nucleic acid molecules encode

   hsa-miR-21-5p、hsa-miR-92a-3p、hsa-miR-124-3p、hsa-miR-184、hsa-miR-196a-5p、hsa-miR-204-5p、hsa-miR-486-5p、hsa-miR-146a-5p、hsa-miR-

   122-5P and/or hsa-miR-150-5p.
10. 10. The use of claim 8, wherein the one or more polynucleic acid molecules are with corresponding mirnas

    The miRNA is miR-21-5p, hsa-miR-92a-3p and hsa-3 p which are partially or completely complementary

    miR-122-5p，hsa-miR-124-3p，hsa-miR-146a-5p，miR-150-5p，hsa-miR-

    184, Hsa-miR-196a-5p, hsa-miR-204-5p and/or hsa-miR-486-5p.