CN110408703B

CN110408703B - Colorectal cancer miRNA marker and application thereof

Info

Publication number: CN110408703B
Application number: CN201910751173.XA
Authority: CN
Inventors: 崔健; 姜薇; 李雪健; 王杰
Original assignee: Hebei Renbo Technology Co ltd
Current assignee: Hebei Renbo Technology Co ltd
Priority date: 2019-08-15
Filing date: 2019-08-15
Publication date: 2022-02-22
Anticipated expiration: 2039-08-15
Also published as: CN110408703A

Abstract

The invention discloses miRNA markers of colorectal cancer, which are hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p, and specifically, the expressions of the markers hsa-miR-3160-3p and hsa-miR-3184-5p are respectively and sequentially reduced and the expression of the marker hsa-miR-770-5p is sequentially increased in blood of patients with colorectal hyperplastic polyps, colorectal adenomas and colorectal adenocarcinoma. Further discloses application of the miRNA marker in preparing a product for diagnosing, prognosing, preventing or treating colorectal cancer. Also disclosed are kits for the diagnosis or prognosis of colorectal cancer, and pharmaceutical compositions for the prevention and/or treatment of colorectal cancer. The miRNA marker can be used for quickly and effectively carrying out early detection on the colorectal cancer, and can also provide a treatment target and an important basis for clinical application such as gene therapy, drug therapy and the like.

Description

Colorectal cancer miRNA marker and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a colorectal cancer miRNA marker and application thereof.

Background

Colorectal cancer (carcinoma of colon and recatum) is a common malignancy in the gastrointestinal tract, with incidence and mortality secondary to gastric, esophageal, and primary liver cancers among digestive malignancies. In recent years, the incidence of colorectal cancer in China tends to increase year by year, the cause of the colorectal cancer is not completely understood, but some factors such as environment, dietary structure, living habits (drinking and smoking) and the like may induce colorectal cancer, and familial polyps, ulcerative colorectal inflammation, familial tumor genetic disease and the like are high-risk factors for colorectal cancer.

Currently, the only effective treatment is surgery, and in advanced colon cancer, its survival rate is also dependent on chemotherapy. Data statistics show that cancer cells have metastasized when more than 57% of patients are diagnosed, whereas 5-year survival rates for early stage colorectal cancer patients can reach 90%, but 5-year overall survival rates for late stage and metastatic patients are only 15%. Therefore, in view of the importance of early diagnosis, the discovery of new diagnostic markers is needed to discover tumors early.

With the rapid development of molecular biology technology and the continuous and deep research of colorectal cancer mechanism, more and more related biomarkers are continuously discovered and reported. From the research progress of non-coding RNA, some miRNA, lncRNA and circRNA have application prospects as disease biomarkers. miRNA is newly discovered non-coding small-molecule RNA with the length of about 18-25 nucleotides, is highly conserved in evolution, accounts for about 1% of the genome, is widely considered to be closely related to the occurrence of human diseases, and provides a new idea for people to know cancer at the gene level. Researches have proved that the abnormal regulation and control of miRNA are closely related to the formation and development of tumor, and the target gene of miRNA is mostly the gene involved in the biological effects of transcription, signal transduction, tumorigenesis, etc. At present, the specific expression of numerous mirnas in colorectal cancer tumor tissues, cell lines and normal tissues has been found.

In summary, the detection of the miRNA marker for colorectal cancer is helpful for solving the problems of timely diagnosis and effective treatment of colorectal cancer.

Disclosure of Invention

In order to realize early diagnosis and intervention of colorectal cancer, the invention aims to provide application of a colorectal cancer miRNA marker in preparation of a product for diagnosis, prognosis, prevention or treatment of colorectal cancer.

The onset of colorectal cancer generally goes through three stages: first from a colorectal hyperplastic polyp to a colorectal adenoma, and then from a colorectal adenoma to a colorectal adenocarcinoma. Therefore, the inventor collects blood samples of patients with colorectal diseases at corresponding stages, analyzes the reasons of the miRNA content difference and the main factors causing the difference by detecting the content of a plurality of miRNAs in the blood samples of the patients at different stages, and selects hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p with the most obvious difference as the targets of further research.

The inventor finds that the expression of the markers hsa-miR-3160-3p and hsa-miR-3184-5p is sequentially reduced and the expression of the marker hsa-miR-770-5p is sequentially increased in the blood of a colorectal hyperplastic polyp patient, a colorectal adenoma patient and a colorectal adenocarcinoma patient respectively. This shows that in the course of the condition of a patient with colorectal hyperplastic polyps deteriorating into colorectal adenomas, and even further progressing into colon adenocarcinoma, hsa-miR-3160-3p and hsa-miR-3184-5p respectively show a tendency of down-regulation of expression in the blood of the patient, while hsa-miR-770-5p shows a tendency of up-regulation of expression in the blood of the patient.

We further validated by qRT-PCR, and further based on mechanistic studies at the cellular level, the role of the mirnas in the prevention and diagnosis of colorectal diseases, in particular colorectal cancer, was established. The experimental result shows that hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p can be used as colorectal cancer markers.

In an embodiment of the invention, the colorectal cancer miRNA markers are one or more of hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p, wherein the expression of the markers hsa-miR-3160-3p and hsa-miR-3184-5p is sequentially reduced and the expression of the markers hsa-miR-770-5p is sequentially increased in the blood of a colorectal hyperplastic polyp patient, a colorectal adenoma patient and a colorectal adenocarcinoma patient, respectively.

The term "upregulated expression" as used herein refers to a sequence of a specific miRNA sequence whose measurement of the amount of the sequence indicates that the level of expression of the gene is increased in a biological sample, such as blood, isolated from a colorectal cancer patient or an individual at risk of having colorectal cancer, as compared to a normal individual. Conversely, "down-regulated expression" refers to a measurement of the amount of a particular miRNA sequence that indicates a decreased level of expression of that gene in a biological sample, such as blood, isolated from a colorectal cancer patient or an individual at risk for colorectal cancer, as compared to a normal individual.

To achieve the above object, the present invention first provides a kit for diagnosis or prognosis of colorectal cancer, comprising primers and instructions for specifically amplifying one or more of the colorectal cancer markers hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5 p.

In the present invention, "prognosis" refers to the process or outcome of a cancer patient after inhibiting or alleviating tumor growth by surgery, chemotherapy, drug treatment, or a combination thereof. Prognosis may be the inhibition or alleviation of the vital state of colorectal cancer by surgery, chemotherapy, drug treatment, or a combination thereof, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 years or more after growth. Prognosis can be assessed by detecting a marker, which is one or more genes. The prognostic assessment may be performed by: determining whether the prognosis of the patient is good, or determining the probability of a good or poor prognosis, based on the presence or absence, or increase or decrease, of the marker.

Further, the primer for specifically amplifying the hsa-miR-3160-3p gene comprises a reverse transcription primer with a sequence of SEQ ID NO. 1 and a cDNA amplification primer pair with sequences of SEQ ID NO. 2 and SEQ ID NO. 3; the primer for specifically amplifying the hsa-miR-3184-5p gene comprises a reverse transcription primer with a sequence of SEQ ID NO. 4 and a cDNA amplification primer pair with sequences of SEQ ID NO. 5 and SEQ ID NO. 6; and the primer for specifically amplifying the hsa-miR-770-5p gene comprises a reverse transcription primer with a sequence of SEQ ID NO. 7 and a cDNA amplification primer pair with a sequence of SEQ ID NO. 8 and a sequence of SEQ ID NO. 9; wherein, the primers for amplifying the internal reference snRNA U6 comprise a reverse transcription primer with a sequence of SEQ ID NO. 10 and a cDNA amplification primer pair with a sequence of SEQ ID NO. 11 and a sequence of SEQ ID NO. 12.

The kit may also include reagents commonly used in PCR reactions,such as reverse transcriptase, buffer, dNTPs, MgCl₂DEPC water and Taq enzyme, etc.; standards and/or controls may also be included.

One aspect of the invention provides application of hsa-miR-3160-3p, hsa-miR-3184-5p or analogues thereof in preparation of a pharmaceutical composition for preventing and/or treating colorectal cancer.

In another aspect, the invention provides an application of the hsa-miR-770-5p inhibitor in preparation of a pharmaceutical composition for preventing and/or treating colorectal cancer.

One aspect of the invention provides a pharmaceutical composition for preventing and/or treating colorectal cancer, wherein the pharmaceutical composition comprises hsa-miR-3160-3p, hsa-miR-3184-5p or an analogue thereof having biological activity, and a pharmaceutically-acceptable carrier.

In yet another aspect, the invention provides a pharmaceutical composition for the prevention and/or treatment of colorectal cancer, wherein the pharmaceutical composition comprises an hsa-miR-770-5p inhibitor, and a pharmaceutically-acceptable carrier; the miRNA inhibitor is a substance capable of inhibiting expression of hsa-miR-770-5p, e.g., a corresponding chemical inhibitor or siRNA, dsRNA, shRNA, miRNA, antisense nucleotide, etc., such as miRNA inhibitor (corresponding chemically modified mature miRNA complementary single strand, ready-to-use) purchased from ribo (Ribobio) inc.

The effective dosage of the hsa-miR-770-5p inhibitor can be adjusted correspondingly according to the administration mode, the severity of the disease to be treated and the like. The preferred effective amount can be determined by one of ordinary skill in the art by combining various factors. Such factors include, but are not limited to: pharmacokinetic parameters of the hsa-miR-770-5p inhibitor, health of the patient being treated, body weight, route of administration, and the like.

In the above aspect of the present invention, the pharmaceutical composition may further comprise other agents that inhibit or treat colorectal cancer.

Advantageous effects

The hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p of the invention have obvious differential expression in the blood of patients at corresponding stages in the three successive development stages of colorectal hyperplastic polyps, colorectal adenomas and colorectal adenocarcinoma of colon cancer patients, so that the hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p can be used as miRNA markers for colorectal cancer diagnosis and prognosis. The diagnostic kit for detecting colorectal cancer provided by the invention can be used for diagnosis and prognosis of colorectal cancer related to hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p, thereby being beneficial to early prevention and/or treatment of the disease. The miRNA disclosed by the invention can be used for quickly and effectively carrying out early detection on colorectal cancer, and provides a treatment target and an important basis for clinical application such as gene therapy, drug therapy and the like.

Drawings

FIG. 1 shows that in the blood of a colorectal hyperplastic polyp patient, a colorectal adenoma patient and a colorectal adenocarcinoma patient, respectively, the expression of the markers hsa-miR-3160-3p and hsa-miR-3184-5p is sequentially reduced, and the expression of the marker hsa-miR-770-5p is sequentially increased.

FIG. 2 shows control HCT116 cells compared to the cell invasiveness of HCT116 cells transfected with an hsa-miR-3160-3p inhibitor, an hsa-miR-3184-5p inhibitor, and an hsa-miR-770-5p mimetic, respectively.

FIG. 3 shows the cell migration ability of control HCT116 cells compared to HCT116 cells transfected with hsa-miR-3160-3p inhibitor, hsa-miR-3184-5p inhibitor and hsa-miR-770-5p mimic, respectively, in a cell-scratch experiment after 48h of culture.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Examples

Example 1 sample miRNA extraction

Blood samples were collected from 10 patients with colorectal hyperplastic polyps, colorectal adenomas and colorectal adenocarcinomas, respectively. The miRNA in the serum of the sample is obtained by a miRNA extraction kit (manufacturer: Tiangen Biochemical technology (Beijing) Co., Ltd.; product batch number: DP 501). The method comprises the following specific steps:

1) adding 900 mul of lysate MZA into every 200 mul of serum, oscillating and uniformly mixing for 30s by an oscillator until the lysate is completely homogenized, and reversing and uniformly mixing;

2) standing at room temperature for 5min to completely separate nucleic acid protein complex;

3) adding chloroform 200 μ l, covering tube cap, shaking the centrifuge tube with force, mixing, and standing at room temperature for 5 min;

4) after centrifugation at 12000rpm for 15min at 4 ℃, the sample will be divided into three layers: a yellow organic phase, a white intermediate layer and a colorless aqueous phase. The RNA is mainly in the water phase, the upper water phase is sucked into another new centrifugal tube, and the protein substance between the two water phases is not sucked;

5) measuring the volume of the transfer solution, slowly adding 3 times of anhydrous ethanol, and mixing (at this time, precipitation may occur). Transferring the obtained solution and the precipitate into an adsorption column miRelute, standing at room temperature for 2min, centrifuging at 12000rpm at room temperature for 30s, carefully removing supernatant, and returning the adsorption column miRelute into a collection tube;

6) adding 700 μ l deproteinized solution MRD (please check whether ethanol is added or not) into the adsorption column miRelute, standing at room temperature for 2min, centrifuging at 12000rpm at room temperature for 30s, carefully discarding supernatant, and returning the adsorption column miRelute to the collection tube;

7) adding 500 μ l of rinsing solution RW (please check whether ethanol is added or not) into the adsorption column miRelute, standing at room temperature for 2min, centrifuging at 12000rpm at room temperature for 30s, carefully discarding supernatant, and returning the adsorption column miRelute to the collection tube;

8) repeating the step 7) once;

9) centrifuging at 12000rpm for 2min at room temperature, discarding the collection tube, and standing the column MiRelute at room temperature for a while to air-dry completely;

10) transferring the adsorption column miRelute into a new RNase-Free 1.5ml centrifuge tube, and adding 15-30 μ l RNase-Free ddH to the center of the adsorption membrane₂O, standing at room temperature for 2min, and centrifuging at 12000rpm at room temperature for 2 min.

Finally, the RNA concentration and purity were measured with a NanoDrop One spectrophotometer and frozen at-70 ℃.

Example 2 validation of expression of hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p by qRT-PCR

1. Reverse transcription to synthesize cDNA

M-MLV reverse transcriptase (promega, cat # 1701), dNTP mixture (dNTP mix) (promega, cat # U1511), random primer (promega, cat # C1181) and RNase inhibitor (promega, cat # N251B) are adopted for cDNA reverse transcription, the experimental operation is carried out according to the product instruction, and the specific operation is as follows:

mu.g of miRNA extracted in example 1 is mixed with 1 mu L of random primer at 70 ℃ for 10 min; subsequently, a reverse transcription buffer, dNTP, a reverse transcriptase inhibitor and M-MLV were added to a 25. mu.L reaction system to perform reverse transcription to synthesize cDNA, and the obtained cDNA sample was diluted 4-fold and stored in a refrigerator at-20 ℃ for future use.

2.Real-Time PCR

2.1 Instrument and analytical method

Using ABI 7500 type fluorescent quantitative PCR instrument, adopting 2^-ΔΔCtThe method carries out relative quantitative analysis of data.

2.2 primer design

On-line primer design software is adopted, and the gene sequence refers to the sequence given by NCBI website: hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p, wherein snRNA U6 is selected as an internal reference, and the primers are synthesized by Shanghai Jie Rui company after being designed. The specific primer sequences are as follows:

TABLE 1 primer sequences

The operation process is as follows:

TABLE 2 Real-Time PCR reaction System

Components	Amount of addition
		2×mix	10μL
Upstream primer (10. mu.M)	1μL
		Downstream primer (10. mu.M)	1μL
Form panel	1μL
		Adding sterilized distilled water	To 20 μ L

By Fast

Green Master Mix (ThermoFisher, cat # 4385612) was amplified with the gene of interest primers and primers for internal reference snRNA U6, respectively. The experimental operation was carried out according to the product instructions. The amplification procedure was: 95 ℃ for 5min, (95 ℃ 15sec, 60 ℃ 20sec, 72 ℃ 20sec) × 40 cycles. At the same time, the dissolution curve analysis is carried out at 60-95 ℃. After the reaction is finished, 5 mul of PCR product is subjected to 2% agarose electrophoresis, miRNA conforming to the size band of the target fragment is amplified again and sequenced, and the result is subjected to sequence comparison by blast software.

3. Results of the experiment

The inflection point of the real-time quantitative PCR (qRT-PCR) amplification curve is clear, the overall parallelism of the amplification curve is good, and the amplification efficiency of each reaction tube is similar; the base line is flat without rising phenomenon, the slope of the exponential phase of the curve is larger, which shows that the amplification efficiency is higher; the dissolution curves of the sample amplification products are all unimodal, which indicates that the amplification productsOnly one strip is specifically amplified; according to the relative quantitative formula of qRT-PCR: 2^-ΔΔCtX 100%, and comparing the expression levels of hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5 p. The results are shown in figure 1, and the expression of the markers hsa-miR-3160-3p and hsa-miR-3184-5p is sequentially reduced and the expression of the marker hsa-miR-770-5p is sequentially increased in the blood of patients with colorectal hyperplastic polyps, colorectal adenomas and colorectal adenocarcinoma respectively.

Example 3 Effect of hsa-miR-3160-3p inhibitor, hsa-miR-3184-5p inhibitor and hsa-miR-770-5p mimetic on migration Performance of HCT116 cells

Transwell migration experiment

1) Preparation of cells

Preparing a six-well plate for culturing control HCT116 cells and HCT116 cells transfected with hsa-miR-3160-3p inhibitor, hsa-miR-3184-5p inhibitor and hsa-miR-770-5p mimic (respectively corresponding miRNA complementary single strands or mimics from Ruibo corporation), pretreating, and culturing overnight (at least 10h) with serum-free medium; then, cell-cell links were broken with 0.025% pancreatin and digestion was stopped with serum-free medium, cell counting was performed, and the total volume of the cell suspension was adjusted so that the number of cells per mL of the medium was approximately the same.

2) Transwell 24-well plate seeded cells

Adding 800 μ L of liquid to each well of the well below the Transwell plate, wherein different chemokines are added; adding a cell suspension with the same volume and the same cell number into a small chamber above a Transwell plate; then culturing in a carbon dioxide incubator for 12-24 h.

3) Dyeing process

Fixing the mixture for 15-20 min by using 4% paraformaldehyde solution at room temperature, and washing the mixture for 2-3 times by using PBS (phosphate buffer solution); then staining with 1% gentian violet dye solution (dissolved in PBS) for about 30min, and washing with PBS; then, the inner surface of the chamber was gently wiped with a cotton swab, taking care not to deform the bottom of the chamber, and washed with PBS.

4) PBS was added dropwise to the slide, and the chamber was placed on PBS, observed under the mirror and photographed.

The results are shown in figure 2, where HCT116 cells transfected with hsa-miR-3160-3p inhibitor, hsa-miR-3184-5p inhibitor and hsa-miR-770-5p mimetic, respectively, penetrated significantly more cells through the membrane into the lower chamber than control HCT116 cells, indicating that the cell invasiveness was significantly increased, resulting in an increased number of HCT116 migrating cells.

2. Cell scratch test:

1) preparing a 12-hole plate, and drawing a straight line with black strokes at the bottom of the hole plate for photographing and positioning after scratching.

2) Control HCT116 cells and HCT116 cells transfected with hsa-miR-3160-3p inhibitor, hsa-miR-3184-5p inhibitor and hsa-miR-770-5p mimic, respectively, are pretreated, and after the cells are overgrown, a scratch perpendicular to the drawn line is made. Washing with PBS, and adding a proper amount of culture medium.

3) The lens was observed under the mirror and photographed at time points 0, 48 h.

Results are shown in fig. 3, similar results appear in the cell scratch experiment after 48h of culture, and compared with control HCT116 cells, HCT116 cells transfected with hsa-miR-3160-3p inhibitor, hsa-miR-3184-5p inhibitor and hsa-miR-770-5p mimic have significantly enhanced repair in the scratch region, indicating that the cell migration capacity is significantly increased.

The above experimental results all show that the hsa-miR-3160-3p inhibitor, the hsa-miR-3184-5p inhibitor and the hsa-miR-770-5p analogue obviously promote the migration of HCT116 cells.

Example 4 kit for detecting colorectal cancer

Based on the primer sequences for RT-PCR listed in example 2 above, kits for detecting colorectal cancer, which include one or more of the following 3 sets of primers, were respectively assembled:

a. the primer for specifically amplifying the hsa-miR-3160-3p gene comprises a reverse transcription primer with a sequence of SEQ ID NO. 1 and a cDNA amplification primer pair with sequences of SEQ ID NO. 2 and SEQ ID NO. 3;

b. the primer for specifically amplifying the hsa-miR-3184-5p gene comprises a reverse transcription primer with a sequence of SEQ ID NO. 4 and a cDNA amplification primer pair with sequences of SEQ ID NO. 5 and SEQ ID NO. 6;

c. the primer for specifically amplifying the hsa-miR-770-5p gene comprises a reverse transcription primer with a sequence of SEQ ID NO. 7 and a cDNA amplification primer pair with a sequence of SEQ ID NO. 8 and a sequence of SEQ ID NO. 9.

Specifically, for example, the following kits:

1. the first kit comprises a primer for specifically amplifying the hsa-miR-3160-3p gene, wherein the primer comprises a reverse transcription primer with a sequence of SEQ ID NO. 1 and a cDNA amplification primer pair with sequences of SEQ ID NO. 2 and SEQ ID NO. 3;

2. the second kit comprises a primer for specifically amplifying the hsa-miR-3184-5p gene, wherein the primer comprises a reverse transcription primer with a sequence of SEQ ID NO. 4 and a cDNA amplification primer pair with sequences of SEQ ID NO. 5 and SEQ ID NO. 6;

3. the third kit comprises a primer for specifically amplifying the hsa-miR-770-5p gene, wherein the primer comprises a reverse transcription primer with a sequence of SEQ ID NO. 7 and a cDNA amplification primer pair with sequences of SEQ ID NO. 8 and SEQ ID NO. 9;

4. the kit IV comprises: the primers for specifically amplifying the hsa-miR-3160-3p gene comprise a reverse transcription primer with a sequence of SEQ ID NO. 1 and a cDNA amplification primer pair with sequences of SEQ ID NO. 2 and SEQ ID NO. 3; and a primer for specifically amplifying the hsa-miR-3184-5p gene, wherein the primer comprises a reverse transcription primer with a sequence of SEQ ID NO. 4 and a cDNA amplification primer pair with sequences of SEQ ID NO. 5 and SEQ ID NO. 6;

5. the fifth kit comprises: the primers for specifically amplifying the hsa-miR-3184-5p gene comprise reverse transcription primers with the sequence of SEQ ID NO. 4 and cDNA amplification primer pairs with the sequences of SEQ ID NO. 5 and SEQ ID NO. 6; and a primer for specifically amplifying the hsa-miR-770-5p gene, wherein the primer comprises a reverse transcription primer with a sequence of SEQ ID NO. 7 and a cDNA amplification primer pair with a sequence of SEQ ID NO. 8 and a sequence of SEQ ID NO. 9;

6. the kit six comprises: the primers for specifically amplifying the hsa-miR-3160-3p gene comprise a reverse transcription primer with a sequence of SEQ ID NO. 1 and a cDNA amplification primer pair with sequences of SEQ ID NO. 2 and SEQ ID NO. 3; and a primer for specifically amplifying the hsa-miR-770-5p gene, wherein the primer comprises a reverse transcription primer with a sequence of SEQ ID NO. 7 and a cDNA amplification primer pair with a sequence of SEQ ID NO. 8 and a sequence of SEQ ID NO. 9;

in addition, the kit also comprises primers for specifically amplifying the internal reference snRNA U6, wherein the primers comprise a reverse transcription primer with a sequence of SEQ ID NO. 10 and a cDNA amplification primer pair with a sequence of SEQ ID NO. 11 and a sequence of SEQ ID NO. 12; and SYBR Green polymerase chain reaction systems, such as PCR buffer, SYBR Green fluorescent dyes, dNTPs. The PCR buffer solution comprises 25mM KCl and 2.5mM MgCl₂，200mM(NH₄)₂SO₄. Through the optimization of the primer concentration and the annealing temperature, the reaction system is finally determined as shown in Table 3:

TABLE 3 PCR reaction System

Components	Amount of addition
		SYBR Green polymerase chain reaction system	10μL
Forward primer (10. mu.M)	1μL
		Reverse primer (10. mu.M)	1μL
Template cDNA	1μL
		Adding sterilized distilled water	To 20 μ L

The optimal reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, (denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 20sec, extension at 72 ℃ for 20sec) x 40 cycles.

For ease of use, the kit may further comprise controls: normal cDNA samples of one or more of the 3 miRNA genes.

Taking a biological sample of a detected person, extracting RNA from the biological sample by using a conventional method (or using a specific kit), using a reagent in the kit, carrying out PCR reaction according to an optimal reaction system and conditions, using normal cDNA in the kit as control cDNA in Real-Time PCR quantitative detection, and measuring the change of the expression quantity of hsa-miR-3160-3p, hsa-miR-3184-5p and/or hsa-miR-770-5p genes in the biological sample of the detected person relative to the expression quantity of the normal cDNA.

The subject may be an individual not diagnosed with colorectal cancer, and the detection result may be used for risk assessment or disease diagnosis and natural prognosis of the individual with a possibility of suffering from colorectal cancer.

The subject can be an individual treated for colorectal cancer, and the detection result can be used for evaluating the curative effect and treating prognosis of the colorectal cancer treatment on the individual.

The kit of the invention detects the expression condition of the specific miRNA marker gene through the specific primer, is convenient to detect, and greatly improves the sensitivity and specificity for diagnosing the colorectal cancer, so that the kit can help to guide early diagnosis and more effective individualized treatment when put into practice.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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Claims

1. The application of the reagent for detecting the miRNA marker in the preparation of colorectal cancer diagnosis products is characterized in that the marker is hsa-miR-3160-3 p.

2. The use according to claim 1, wherein the expression of marker hsa-miR-3160-3p is decreased in the blood of patients with colorectal hyperplastic polyps, colorectal adenomas and colorectal adenocarcinomas, respectively, in a sequential manner.

Application of hsa-miR-3160-3p or analogues thereof in preparation of pharmaceutical compositions for preventing and/or treating colorectal cancer.