CN110408703A

CN110408703A - Colorectal cancer miRNA marker and its application

Info

Publication number: CN110408703A
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: 2019-11-05
Anticipated expiration: 2039-08-15
Also published as: CN110408703B

Abstract

The invention discloses the miRNA markers of colorectal cancer, the marker is hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p, specifically, respectively in the blood of Colon and rectum hyperplastic polyp patient, Colon and rectum adenoma patients and Colon and rectum gland cancer patient, the expression of the marker hsa-miR-3160-3p and hsa-miR-3184-5p successively reduces respectively, and the expression of the marker hsa-miR-770-5p successively increases.And further disclose application of the miRNA marker in the diagnosis of preparation colorectal cancer, prognosis, prevention or treatment product.Also disclose the kit for diagnosis of colorectal carcinoma or prognosis, and prevention and/or the pharmaceutical composition for treating colorectal cancer.Early detection fast and effeciently can be carried out to colorectal cancer using miRNA marker of the present invention, moreover it is possible to provide therapy target and important evidence for clinical applications such as gene therapy, drug therapies.

Description

Colorectal cancer miRNA marker and its application

Technical field

The present invention relates to field of biotechnology, and in particular to colorectal cancer miRNA marker and its application.

Background technique

Colorectal cancer (carcinoma of colon and rectum) is malignant tumour common in gastrointestinal tract, hair Sick rate and case fatality rate are only second to gastric cancer, the cancer of the esophagus and primary carcinoma of liver in alimentary system malignant tumour.In recent years, colorectal cancer Disease incidence in China shows an increasing trend year by year, though the cause of disease is not fully apparent from, some factors for example environment, dietary structure, Living habit (smoking of drinking) etc. may induce colorectal cancer, in addition, familial polyposis, ulcerative colitis and familial Tumorgenesis medical history etc. is also the high risk factor that colorectal cancer occurs.

Currently, the only effective therapeutic modality is operation, and its survival rate is also relied on chemotherapy in advanced colon cancer.Number Be more than according to statistics when 57% patient makes a definite diagnosis cancer cell shifted, and 5 years survival rates of early stage colorectal cancer patients can reach 90%, But advanced stage and metastatic patient 5 years overall survivals are only 15%.Therefore, it in view of the importance of early diagnosis, needs to find New diagnosis marker carrys out early detection tumour.

With the fast development of Protocols in Molecular Biology and deepening continuously for colorectal cancer Mechanism Study, more and more phases Biomarker is closed constantly to be found and reported.From the point of view of the progress of non-coding RNA, some miRNA, lncRNA, CircRNA has the application prospect as disease biomarkers.MiRNA is that latest find is about 18-25 nucleotide Non-coding microRNA, upper highly conserved evolving, quantity accounts for about the 1% of genome, has generally believed miRNA and the mankind Disease has close connection, is found to be people in gene level understanding cancer and provides new approaches.Research has proven to The Abnormal regulation of miRNA and tumour formed and be in progress it is closely related, the target gene majority of miRNA be participate in transcription, signal transduction, The gene of the biological effects such as tumour generation.At present, it has been found that in colorectal cancer tumor tissues, cell line and normal tissue There is the specifically expressing of numerous miRNA.

It diagnoses and has in time in conclusion will be helpful to solution colorectal cancer by detection colorectal cancer miRNA marker The problem of effect treatment aspect.

Summary of the invention

In order to realize the early diagnosis and intervention of colorectal cancer, the purpose of the present invention is to provide colorectal cancer miRNA marks Application of the will object in the diagnosis of preparation colorectal cancer, prognosis, prevention or treatment product.

Colorectal Cancer process generally understands experienced three stages: being first Colon and rectum by the development of Colon and rectum hyperplastic polyp Then adenoma is Colon and rectum gland cancer by the development of Colon and rectum adenoma.For this purpose, the present inventor, which acquires colorectal diseases patient, corresponds to the stage Blood sample, by detection different phase blood samples of patients sample in a variety of miRNA content, analyze the miRNA content difference The reason of and lead to the principal element of the difference, we select wherein difference most significant hsa-miR-3160-3p, hsa-miR- 3184-5p and hsa-miR-770-5p is as the target further studied.

The inventors discovered that suffering from respectively in Colon and rectum hyperplastic polyp patient, Colon and rectum adenoma patients and Colon and rectum gland cancer In the blood of person, the expression of the marker hsa-miR-3160-3p and hsa-miR-3184-5p successively reduces respectively, and institute The expression for stating marker hsa-miR-770-5p successively increases.Which show be in the deterioration of Colon and rectum hyperplastic polyp patient's patient's condition Colon and rectum adenoma, or even during being further development of adenocarcinoma of colon, hsa-miR-3160-3p and hsa-miR-3184-5p The trend that expression is presented respectively and lowers in blood samples of patients, and then presentation expression of the hsa-miR-770-5p in blood samples of patients The trend of up-regulation.

We are further verified by qRT-PCR, then the Mechanism Study based on cellular level, specify that the miRNA exists Effect in prevention and diagnosis colorectal diseases, especially colorectal cancer.It is of the invention the experimental results showed that, hsa-miR- 3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p can be used as colorectal cancer marker.

In embodiments of the invention, the colorectal cancer miRNA marker is hsa-miR-3160-3p, hsa- One or more of miR-3184-5p and hsa-miR-770-5p, wherein straight in Colon and rectum hyperplastic polyp patient, knot respectively In the blood of enteric adenoma patient and Colon and rectum gland cancer patient, the marker hsa-miR-3160-3p and hsa-miR-3184-5p Expression successively reduce respectively, the expression of the marker hsa-miR-770-5p successively increases.

Terms used herein " expression up-regulation " refers to that for specific miRNA sequence, the measurement of sequence amount shows and normal Body ratio, in colorectal cancer patients or the biological sample such as blood that are separated with the individual for suffering from Risk of Colorectal Cancer, the gene Expression increases.Conversely, " expression is lowered " refers to that, for specific miRNA sequence, the measurement of sequence amount shows and normal individual Compare, from colorectal cancer patients or with suffer from Risk of Colorectal Cancer individual separation biological sample such as blood in, the gene Expression reduce.

To achieve the above object, present invention firstly provides the kit for diagnosis of colorectal carcinoma or prognosis, it includes spies One in colorectal cancer marker hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p described in specific amplification A or multiple primer and specification.

In the present invention, " prognosis " refers to cancer patient by suppressions such as operation, chemotherapy, drug therapy or combinations thereof processing System alleviates process or result after tumour growth.Prognosis, which can be to handle by operation, chemotherapy, drug therapy or combinations thereof, to be pressed down Life state when 1,2,3,4,5,6,7,8,9,10,15,20 year or more long after system or alleviation colorectal cancer growth.Prognosis can be with It is assessed by detection marker, the marker is one or more genes.Prognosis evaluation can be performed such that according to mark Object with or without or being raised and lowered, determine whether the prognosis of patient good, or determine good prognosis or poor prognosis Probability.

Further, it is SEQ ID NO:1 that the primer of the specific amplification hsa-miR-3160-3p gene, which includes sequence, Reverse transcription primer and sequence be SEQ ID NO:2 and SEQ ID NO:3 cDNA amplimer pair；The specificity expands The primer of increasing hsa-miR-3184-5p gene includes the reverse transcription primer that sequence is SEQ ID NO:4 and sequence is SEQ ID The cDNA amplimer pair of NO:5 and SEQ ID NO:6；And the primer of the specific amplification hsa-miR-770-5p gene Expand including the cDNA that the reverse transcription primer that sequence is SEQ ID NO:7 and sequence are SEQ ID NO:8 and SEQ ID NO:9 Increase primer pair；Wherein, the primer for expanding internal reference snRNA U6 includes reverse transcription primer of the sequence for SEQ ID NO:10, Yi Jixu It is classified as the cDNA amplimer pair of SEQ ID NO:11 and SEQ ID NO:12.

The kit can also include that PCR reacts common agents, such as reverse transcriptase, buffer, dNTPs, MgCl₂、 DEPC water and Taq enzyme etc.；Standard items and/or reference substance can also be contained.

It is pre- in preparation that one aspect of the invention provides hsa-miR-3160-3p, hsa-miR-3184-5p or its analog Application in anti-and/or treatment colorectal cancer pharmaceutical composition.

Another aspect of the invention provides hsa-miR-770-5p inhibitor in preparation prevention and/or treatment colorectal cancer Pharmaceutical composition in application.

One aspect of the invention provides prevention and/or treats the pharmaceutical composition of colorectal cancer, wherein the medicine group Closing object includes hsa-miR-3160-3p, hsa-miR-3184-5p or the analog with its bioactivity, and pharmaceutically may be used The carrier of receiving.

Another aspect of the present invention provides prevention and/or treats the pharmaceutical composition of colorectal cancer, wherein the drug Composition includes hsa-miR-770-5p inhibitor and pharmaceutically acceptable carrier；The miRNA inhibitor is that can press down The substance of hsa-miR-770-5p processed expression, for example, corresponding chemical inhibitor or siRNA, dsRNA, shRNA, miRNA, anti- (the mature miRNA of corresponding chemical modification is mutual from the miRNA inhibitor of sharp rich (Ribobio) company for adopted nucleotide etc., such as purchase Mend single-stranded, instant).

The effective dose of hsa-miR-770-5p inhibitor of the invention can be with the mode and disease to be treated of administration Severity etc. be adjusted correspondingly.It is preferred it is a effective amount of can be each because usually by those of ordinary skill in the art's synthesis It determines.The factor includes but is not limited to: the pharmacokinetic parameter of hsa-miR-770-5p inhibitor, treated patient Health status, weight, administration route etc..

In aforementioned aspect of the present invention, described pharmaceutical composition can also be comprising other of inhibition or treatment colorectal cancer Medicament.

Beneficial effect

Hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p of the invention is in colorectal cancer patients In successively three developing stage of Colon and rectum hyperplastic polyp, Colon and rectum adenoma and Colon and rectum gland cancer, respective stage patient's It is clearly present differential expression in blood, therefore may be used as the miRNA marker of diagnosis of colorectal carcinoma and prognosis.The present invention provides For detecting the diagnostic kit of colorectal cancer, can be used for hsa-miR-3160-3p, hsa-miR-3184-5p and hsa- The diagnosis and prognosis of the relevant colorectal cancer of miR-770-5p, to help that the disease is prevented and/or treated as early as possible. MiRNA of the present invention can not only be used to fast and effeciently to colorectal cancer carry out early detection, and for gene therapy, The clinical applications such as drug therapy provide therapy target and important evidence.

Detailed description of the invention

Fig. 1 is shown respectively in Colon and rectum hyperplastic polyp patient, Colon and rectum adenoma patients and Colon and rectum gland cancer patient In blood, the expression of the marker hsa-miR-3160-3p and hsa-miR-3184-5p successively reduces respectively, the mark The expression of object hsa-miR-770-5p successively increases.

Fig. 2 show control HCT116 cell, has hsa-miR-3160-3p inhibitor, hsa-miR- with transfection respectively The cell invasion power of the HCT116 cell of 3184-5p inhibitor and hsa-miR-770-5p analogies compares.

Fig. 3 is shown after cultivating 48h in cell scratch experiment, compares HCT116 cell, has hsa- with transfection respectively The HCT116 cell of miR-3160-3p inhibitor, hsa-miR-3184-5p inhibitor and hsa-miR-770-5p analogies it is thin Born of the same parents' transfer ability compares.

Specific embodiment

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

Embodiment

Embodiment 1: sample miRNA is extracted

The blood sample of Colon and rectum hyperplastic polyp patient, Colon and rectum adenoma patients and Colon and rectum gland cancer patient are collected respectively Each 10.Pass through miRNA extracts kit (manufacturer: TIANGEN Biotech (Beijing) Co., Ltd.；Batch number: DP501) Obtain the miRNA in the serum of the sample.Specific step is as follows:

1) 900 μ l lysate MZA will be added in every 200 μ l serum, oscillator oscillation mixes 30s to complete homogenate, overturns It mixes；

2) it is stored at room temperature 5min, is kept completely separate nucleic acid-protein compound；

3) chlorination imitates 200 μ l, covers pipe lid, with forced oscillation centrifuge tube, mixes well, be stored at room temperature 5min；

4) 4 DEG C, after 12000rpm centrifugation 15min, sample can divide three layers: the organic phase of yellow, white middle layer and nothing The water phase of color.RNA mainly in water phase, draws upper strata aqueous phase into another new centrifuge tube, is careful not to be drawn onto two layers of water phase Between protein substance；

5) volume for measuring transfer liquid, is slowly added to the dehydrated alcohol of transfer 3 times of volumes of liquid, and mixing (is possible to out at this time It now precipitates).Obtained solution and precipitating are transferred to adsorption column miRelute together, are placed at room temperature for 2min, at room temperature 12000rpm Supernatant is carefully discarded after centrifugation 30s, adsorption column miRelute is put back in collecting pipe；

6) 700 μ l protein liquid removal MRD (please first check whether and ethyl alcohol has been added), room temperature are added into adsorption column miRelute 2min is placed, 12000rpm carefully discards supernatant after being centrifuged 30s at room temperature, and adsorption column miRelute is put back in collecting pipe；

7) 500 μ l rinsing liquid RW (please first check whether and ethyl alcohol has been added) are added into adsorption column miRelute, room temperature is put 2min is set, 12000rpm carefully discards supernatant after being centrifuged 30s at room temperature, and adsorption column miRelute is put back in collecting pipe；

8) it is primary to repeat step 7)；

9) 12000rpm is centrifuged 2min at room temperature, discards collecting pipe, and attached column miRelute is being placed at room temperature for a moment to fill Divide and dries；

10) adsorption column miRelute is transferred in a new RNase-Free 1.5ml centrifuge tube, to absorption center membrane Position adds 15-30 μ l RNase-Free ddH₂O is placed at room temperature for 2min, and 12000rpm is centrifuged 2min at room temperature.

Finally, being frozen with NanoDrop One spectrophotometer measurement RNA concentration and purity in -70 DEG C.

Embodiment 2: hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770- are verified by qRT-PCR The expression of 5p

1. reverse transcription synthesizes cDNA

Using M-MLV reverse transcriptase (promega, article No. 1701), dNTP mixture (dNTP mix) (promega, article No. U1511), random primer (promega, article No. C1181), RNase inhibitor (promega, article No. N251B) carry out cDNA reversion Record, experimental implementation are carried out by product description, and concrete operations are as follows:

First extracted miRNA in l μ g embodiment 1 is mixed with 1 μ L random primer, 70 DEG C, 10min；It then will reversion Record buffer, dNTP, reverse transcriptase inhibitors, M-MLV are added to 25 μ L reaction systems, carry out reverse transcription and synthesize cDNA, will obtain The cDNA sample obtained dilutes 4 times, and it is spare to be then stored in -20 DEG C of refrigerators.

2.Real-Time PCR

2.1 instruments and analysis method

With 7500 type fluorescence quantitative PCR instrument of ABI, using 2^-ΔΔCtMethod carries out data relative quantitative assay.

2.2 design of primers

Using online primer-design software, sequence that gene order is provided referring to the website NCBI: hsa-miR-3160-3p, Hsa-miR-3184-5p and hsa-miR-770-5p, interior participation in the election snRNA U6 are synthesized by Shanghai JaRa company after design of primers. Specific primer sequence is as follows:

1 primer sequence of table

Operating process is as follows:

2 Real-Time PCR reaction system of table

Component	Additional amount
		2×mix	10μL
Upstream primer (10 μM)	1μL
		Downstream primer (10 μM)	1μL
Template	1μL
		Sterile purified water is added	To 20 μ L

Use FastGreen Master Mix (ThermoFisher, article No. 4385612) uses purpose base respectively Because the primer of primer and internal reference snRNA U6 is expanded.Experimental implementation is carried out by product description.Amplification program are as follows: 95 DEG C 5min, (95 DEG C of 15sec, 60 DEG C of 20sec, 72 DEG C of 20sec) × 40 circulations.Simultaneously in 60-95 DEG C of progress solubility curve analysis. After reaction, it takes the PCR product of 5 μ l to carry out 2% agarose electrophoresis, the miRNA of target fragment size strip will be met again It expands and is sequenced, as a result carry out sequence alignment with blast software.

3. experimental result

Real-time quantitative PCR (qRT-PCR) amplification curve inflection point understands that amplification curve entirety collimation is good, shows each reaction The amplification efficiency of pipe is close；Baseline is put down without the phenomenon that raises up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample Amplified production solubility curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the opposite of qRT-PCR Quantitative equation: 2^-ΔΔCt× 100%, compare the table of hsa-miR-3160-3p, hsa-miR-3184-5p and hsa-miR-770-5p Up to level.As a result as shown in Figure 1, suffering from respectively in Colon and rectum hyperplastic polyp patient, Colon and rectum adenoma patients and Colon and rectum gland cancer In the blood of person, the expression of the marker hsa-miR-3160-3p and hsa-miR-3184-5p successively reduces respectively, described The expression of marker hsa-miR-770-5p successively increases.

Embodiment 3:hsa-miR-3160-3p inhibitor, hsa-miR-3184-5p inhibitor and hsa-miR-770-5p mould Quasi- influence of the object to HCT116 cell migration performance

1.Transwell migration experiment

1) prepare cell

Preparing six orifice plates has hsa-miR-3160-3p inhibition for cultivating control HCT116 cell and transfecting respectively Agent, hsa-miR-3184-5p inhibitor and hsa-miR-770-5p analogies (are respectively purchased from the correspondence miRNA of Rui Bo company Complementary single strand or analogies) HCT116 cell, after being pre-processed, change serum free medium overnight incubation (at least 10h)；So Afterwards, intercellular link is interrupted with 0.025% pancreatin, and terminates digestion process with serum free medium, carry out cell count, Adjustment cell suspension total volume keeps every mL culture medium cell number roughly the same.

2) 24 orifice plate inoculating cell of Transwell

800 μ L liquid are added in the every hole of aperture below Transwell plate, wherein adding different chemotactic factor (CF)s；In The cell suspension of the cell numbers such as isometric is added in Transwell plate top chamber；Then in carbon dioxide incubator culture 12 ~for 24 hours.

3) it dyes

15~20min is fixed with 4% paraformaldehyde solution room temperature, PBS is cleaned 2~3 times；It is (molten with 1% gentian violet solution again In PBS) dyeing about 30min, PBS cleaning；Then, small indoor surface is gently wiped with cotton swab, pays attention to not being deformed cell bottom, PBS cleaning.

4) PBS is added dropwise on glass slide, cell is placed on PBS, under the microscope and takes pictures.

As a result as shown in Fig. 2, transfection has hsa-miR-3160-3p inhibitor, hsa- respectively compared to control HCT116 cell The cell that the HCT116 cell-penetrating film of miR-3184-5p inhibitor and hsa-miR-770-5p analogies enters lower room is obvious More, show that the cell invasion power obviously increases, so that HCT116 migrating cell quantity increases.

2. cell scratch experiment:

1) prepare 12 orifice plates, in orifice plate bottom black stroke straight line, in case positioning of taking pictures after scratch.

2) control HCT116 cell and respectively transfection there are into hsa-miR-3160-3p inhibitor, hsa-miR-3184-5p The HCT116 cell of inhibitor and hsa-miR-770-5p analogies is pre-processed, and after cell covers with, is done and has been finished The perpendicular scratch of straight line.PBS cleaning, adds appropriate culture medium.

3) under the microscope and take pictures, time point 0,48h.

As a result thin compared to control HCT116 as shown in figure 3, there is analog result in cell scratch experiment after cultivating 48h Born of the same parents, transfection has hsa-miR-3160-3p inhibitor, hsa-miR-3184-5p inhibitor and hsa-miR-770-5p simulation respectively The HCT116 cell of object is repaired in scratch area and is significantly increased, and shows that the cell migration ability obviously increases.

Above-mentioned experimental result shows hsa-miR-3160-3p inhibitor, hsa-miR-3184-5p inhibitor and hsa- MiR-770-5p analogies have been obviously promoted HCT116 cell migration.

Embodiment 4: for detecting the kit of colorectal cancer

The listed primer sequence for being used for RT-PCR in 2 based on the above embodiment, assembles respectively for detecting colorectal cancer Kit, the kit include one or more groups of in following 3 groups of primers:

A. the primer of specific amplification hsa-miR-3160-3p gene draws including the reverse transcription that sequence is SEQ ID NO:1 Object and sequence are the cDNA amplimer pair of SEQ ID NO:2 and SEQ ID NO:3；

B. the primer of specific amplification hsa-miR-3184-5p gene draws including the reverse transcription that sequence is SEQ ID NO:4 Object and sequence are the cDNA amplimer pair of SEQ ID NO:5 and SEQ ID NO:6；

C. the primer of specific amplification hsa-miR-770-5p gene draws including the reverse transcription that sequence is SEQ ID NO:7 Object and sequence are the cDNA amplimer pair of SEQ ID NO:8 and SEQ ID NO:9.

Specifically, such as following kit:

1. kit one includes the primer of specific amplification hsa-miR-3160-3p gene, the primer includes that sequence is The reverse transcription primer and sequence of SEQ ID NO:1 is the cDNA amplimer pair of SEQ ID NO:2 and SEQ ID NO:3；

2. kit two includes the primer of specific amplification hsa-miR-3184-5p gene, the primer includes that sequence is The reverse transcription primer and sequence of SEQ ID NO:4 is the cDNA amplimer pair of SEQ ID NO:5 and SEQ ID NO:6；

3. kit three includes the primer of specific amplification hsa-miR-770-5p gene, the primer includes that sequence is The reverse transcription primer and sequence of SEQ ID NO:7 is the cDNA amplimer pair of SEQ ID NO:8 and SEQ ID NO:9；

4. kit four includes: the primer of specific amplification hsa-miR-3160-3p gene, the primer includes that sequence is The reverse transcription primer and sequence of SEQ ID NO:1 is the cDNA amplimer pair of SEQ ID NO:2 and SEQ ID NO:3；With And the primer of specific amplification hsa-miR-3184-5p gene, the primer include that the reverse transcription that sequence is SEQ ID NO:4 is drawn Object and sequence are the cDNA amplimer pair of SEQ ID NO:5 and SEQ ID NO:6；

5. kit five includes: the primer of specific amplification hsa-miR-3184-5p gene, the primer includes that sequence is The reverse transcription primer and sequence of SEQ ID NO:4 is the cDNA amplimer pair of SEQ ID NO:5 and SEQ ID NO:6；With And the primer of specific amplification hsa-miR-770-5p gene, the primer include that the reverse transcription that sequence is SEQ ID NO:7 is drawn Object and sequence are the cDNA amplimer pair of SEQ ID NO:8 and SEQ ID NO:9；

6. kit six includes: the primer of specific amplification hsa-miR-3160-3p gene, the primer includes that sequence is The reverse transcription primer and sequence of SEQ ID NO:1 is the cDNA amplimer pair of SEQ ID NO:2 and SEQ ID NO:3；With And the primer of specific amplification hsa-miR-770-5p gene, the primer include that the reverse transcription that sequence is SEQ ID NO:7 is drawn Object and sequence are the cDNA amplimer pair of SEQ ID NO:8 and SEQ ID NO:9；

In addition, the kit further includes the primer of specific amplified internal reference snRNA U6, the primer includes that sequence is SEQ The reverse transcription primer and sequence of ID NO:10 is the cDNA amplimer pair of SEQ ID NO:11 and SEQ ID NO:12；With And SYBR Green polymerase chain reaction system, such as PCR buffer, SYBR Green fluorescent dye, dNTPs.The PCR is slow The ingredient of fliud flushing is 25mM KCl, 2.5mM MgCl₂, 200mM (NH₄)₂SO₄.By to the excellent of primer concentration and annealing temperature Change, final to determine that reaction system is as shown in table 3:

3 PCR reaction system of table

Component	Additional amount
		SYBR Green polymerase chain reaction system	10μL
Forward primer (10 μM)	1μL
		Reverse primer (10 μM)	1μL
Template cDNA	1μL
		Sterile purified water is added	To 20 μ L

Optimum reaction condition are as follows: 95 DEG C of initial denaturation 5min, (95 DEG C of denaturation 15sec, 60 DEG C of annealing 20sec, 72 DEG C extend 20sec) × 40 circulation.

For convenience of use, kit also may include control: one or more normal cDNA in above-mentioned 3 kinds of miR-96 genes Sample.

Subject's biological sample is taken, is extracted from biological sample using conventional method (or using specific kit) RNA is carried out PCR with condition according to optimal reaction system and is reacted using the seminal plasma fructose detection kit, using normal in kit CDNA measures hsa-miR-3160- in subject's biological sample as the control cDNA in Real-Time PCR quantitative detection The expression quantity of the relatively normal cDNA of the expression quantity of 3p, hsa-miR-3184-5p and/or hsa-miR-770-5p gene changes.

Subject can be the individual without diagnosis of colorectal carcinoma, and testing result can be used for carrying out the individual suffering from knot straight The risk assessment or medical diagnosis on disease of intestinal cancer possibility and Nature prognosis.

Subject can be the individual through treatment of colorectal cancer, and testing result can be used for carrying out colorectal cancer to the individual The curative effect evaluation for the treatment of and treatment prognosis.

Kit of the invention detects the expression of specific miRNA marker gene, inspection by the primer of specificity It is convenient to survey, and substantially increases the sensibility and specificity of diagnosis colorectal cancer, therefore this kit is put into and is practiced, can help Guidance early diagnosis and more effective individualized treatment.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

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Claims

1. detecting the miRNA marker of colorectal cancer, which is characterized in that the marker is hsa-miR-3160-3p, hsa- One of miR-3184-5p and hsa-miR-770-5p or a variety of.

2. miRNA marker as described in claim 1, which is characterized in that straight in Colon and rectum hyperplastic polyp patient, knot respectively In the blood of enteric adenoma patient and Colon and rectum gland cancer patient, the marker hsa-miR-3160-3p and hsa-miR-3184-5p Expression successively reduce respectively, the expression of the marker hsa-miR-770-5p successively increases.

3. colorectal cancer miRNA marker described in claim 1 is in diagnosis, prognosis, prevention or the treatment of preparation colorectal cancer Application in product.

4. for diagnosis of colorectal carcinoma or the kit of prognosis, which is characterized in that described in claim 1 comprising specific amplification The primer and specification of colorectal cancer miRNA marker.

5. kit as claimed in claim 4, which is characterized in that the specific amplification hsa-miR-3160-3p gene Primer includes the reverse transcription primer that sequence is SEQ ID NO:1 and sequence is SEQ ID NO:2 and SEQ ID NO:3 CDNA amplimer pair；The primer of the specific amplification hsa-miR-3184-5p gene includes that sequence is SEQ ID NO:4 Reverse transcription primer and sequence are the cDNA amplimer pair of SEQ ID NO:5 and SEQ ID NO:6；The specific amplification The primer of hsa-miR-770-5p gene includes the reverse transcription primer that sequence is SEQ ID NO:7 and sequence is SEQ ID The cDNA amplimer pair of NO:8 and SEQ ID NO:9.

6.hsa-miR-3160-3p, hsa-miR-3184-5p or its analog be in preparation prevention and/or treatment colorectal cancer Application in pharmaceutical composition.

Application of the 7.hsa-miR-770-5p inhibitor in the pharmaceutical composition of preparation prevention and/or treatment colorectal cancer.

8. the pharmaceutical composition of prevention and/or treatment colorectal cancer, which is characterized in that described pharmaceutical composition includes hsa-miR- 3160-3p, hsa-miR-3184-5p or analog and pharmaceutically acceptable carrier with its bioactivity.

9. the pharmaceutical composition of prevention and/or treatment colorectal cancer, which is characterized in that described pharmaceutical composition includes hsa-miR- 770-5p inhibitor and pharmaceutically acceptable carrier.

10. pharmaceutical composition as claimed in claim 8 or 9, which is characterized in that described pharmaceutical composition also includes to inhibit or control Treat other medicaments of colorectal cancer.