CN110438227A

CN110438227A - Purposes of the miR-17-92 in diagnosis of colorectal carcinoma

Info

Publication number: CN110438227A
Application number: CN201910689493.7A
Authority: CN
Inventors: 王淋立; 何敏仪; 莫健; 陈帝武; 王萍
Original assignee: Guangzhou Zhongyu Medical Biotechnology Co Ltd
Current assignee: Guangzhou Zhongyu Medical Biotechnology Co Ltd
Priority date: 2019-07-29
Filing date: 2019-07-29
Publication date: 2019-11-12

Abstract

The invention belongs to field of biotechnology, and in particular to purposes of the miR-17-92 in diagnosis of colorectal carcinoma.Present invention firstly discovers that hypomethylation of the former cancer non-coding RNA miR-17-92 cluster in colorectal cancer is horizontal, it is related to the occurrence and development of colorectal cancer.MiR-17-92 cluster methylation level can be used as the Biological indicators of lesion detection, and auxiliary diagnosis colorectal cancer improves detection specificity, susceptibility, reduces error rate, have highly important Clinical significance of MG to understand Tumorigenesis and diagnosis.

Description

Purposes of the miR-17-92 in diagnosis of colorectal carcinoma

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of purposes of miR-17-92 in diagnosis of colorectal carcinoma.

Background technique

Colorectal cancer (Colorectal Cancer, CRC) is one of high-incidence cancer in the whole world.The morbidity of China's colorectal cancer Rate is about 14.2/100,000, in trend is substantially increased, has become disease incidence and rises most fast one of malignant tumour within nearly 20 years. The generation of colorectal cancer will pass through normal mucosa, hyperplasia, and adenoma is formed, and adenoma canceration is developed up to the interim of infiltration metastasis, Its development process was for more than 10 years.Wherein, adenomatous polyp is the most important precancerous lesion of colorectal cancer.Adenomatous polyp by It is cumulative big, time several years is usually needed to final generation canceration.If colorectal cancer can be in early detection, underwent operative excision Afterwards, cure rate is very high, up to 90% or more.

Colorectal cancer is considered as being accumulated by the multistage multi-step that many factors such as environment, heredity participate at present Pathogenic course, occurs and pathogenic mechanism is rather complicated.Epigenetic (epigenntics) regulates and controls the morbidity in colorectal cancer It is played an important role in journey, i.e., in the case that base sequence does not change in genomic DNA, passes through DNA and histone Modification come controlling gene expression.

Many diseases such as DNA methylation and cancer, aging, senile dementia are closely related.Promoter DNA methylation can lead to Silenced gene transcription makes the expression such as tumor suppressor gene, cell cycle-regulatory gene, apoptogene extremely reduce or not express.Go first Baseization can then induce reactivating and expressing for proto-oncogene, participate in the occurrence and development of tumour.It is previous a large number of studies show that, cancer The methylation level of related gene DNA, which changes, to be carried out methylation level detection to it earlier than malignant hyperplasia and can be used for Prediction, diagnosis, classification and the prognosis evaluation of tumour.Therefore, the methylation level research of cancer related gene DNA promoter, Have become the important research content of epigenetics and epigenomics in tumor area.

The research of Microrna (miRNA) is gradually taken seriously in recent years, becomes the hot spot of tumor research.MiRNA is one The endogenous non-protein that class length is about 22nt encodes single-stranded microRNA, is widely present in eucaryote, it can be with it 3 ' ends non-translational region (3 ' area-UTR) of the mRNA (mRNA) of corresponding target genes combine, to make mRNA that degradation or suppression occur Make its translation.Research shows that miRNA is close with relation between tumor, the role of oncogene or tumor suppressor gene is served as, in tumour cell Growth, proliferation, differentiation, apoptosis, pernicious mesenchymal transformation etc. play a significant role.

MiR-17-92 cluster is also known as C13orf25, is located at No. 13 chromosome of the mankind.MiR-17- is thought in most of researchs 92 clusters are potent proto-oncogenes, are the Typical Representatives of proto-oncogene miRNA.The cluster is located at (the miR-17-92 cluster place MIR17HG Key-gene) in introne, primary transcription is originally the polycistron for being about 0.8kb, 6 independent maturation miRNA are encoded to, That is miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1.They are in embryonic development, siberian crabapple System, kidney and heart development, aging and tumour play a role in occurring.The malignant tumour of various hemopoietic systems, lung cancer and The expression of miR-17-92 cluster height is found in a variety of entity tumors.In Colon and rectum adenoma is developed to colorectal cancer, miR-17-92 cluster Expression quantity significant changes occur, and it is related with the occurrence and development of Colon and rectum adenoma to gland cancer.However, at present for colorectal cancer The research of middle miRNA methylation is less.Effect of the miR-17-92 cluster methylation level in tumour does not have been reported that also.Therefore, it visits The relationship of miR-17-92 cluster methylation level and tumour is begged for, there is highly important face to understand Tumorigenesis and diagnosis Bed directive significance.

Summary of the invention

The present invention is directed to provide a kind of purposes of miR-17-92 in diagnosis of colorectal carcinoma.

Therefore, one of the objects of the present invention is to provide a kind of miR-17-92 cluster methylation sites is making as marker Purposes in standby diagnosis of colorectal carcinoma reagent.

Another object of the present invention is to provide a kind of reagents of the miR-17-92 cluster methylation in detection sample to be tested to exist Prepare the purposes in diagnosis of colorectal carcinoma reagent.

The technical solution adopted in the present invention is as described below.

Detect use of the reagent of the sequence methylation level to be measured in sample to be tested in preparation diagnosis of colorectal carcinoma reagent On the way, the sequence to be measured is located at miR-17-92 cluster transcription initiation site upstream 3.5kb sequence between the 2kb sequence of downstream.

Further, the diagnosis of colorectal carcinoma reagent for carry out diagnosis of colorectal carcinoma, Risk of Colorectal Cancer assessment and/ Or treatment of colorectal cancer recruitment evaluation.

Further, the miR-17-92 cluster is located on human chromosomal 13q31.3, GI:387942364, Gene ID For 407975GRCh38.MiR-17-92 cluster is located on No. 13 chromosome of the mankind, the miR-17-92 recorded in database at present Cluster GI:387942364, Gene ID is 407975GRCh38；The purpose of the present invention is detection miR-17-92 cluster methylations to be The horizontal of methylation of miR-17-92 cluster transcription initiation site upstream 3.5kb sequence to downstream 2kb sequence changes to diagnose knot The generation of the carcinoma of the rectum, therefore even if follow-up data qurush grade, miR-17-92 cluster record information changes, still real with the present invention Matter is identical, without departing from protection scope of the present invention.

Further, the sequence to be measured is located at miR-17-92 cluster transcription initiation site upstream 1.5kb sequence to downstream Between 1.5kb sequence, it is preferable that the sequence to be measured is the reverse complementary sequence of SEQ ID NO:25 or SEQ ID NO:25.

Further, the sequence to be measured may be located on having on the miR-17-92 cluster transcription initiation site of SNP site 3.5kb sequence is swum between the 2kb sequence of downstream.

Further, the sample to be tested is selected from least one of excrement, blood and biopsy.Preferably, described Sample to be tested is excrement.

Further, the method for detecting the sequence methylation level to be measured is selected from pyrosequencing, bisulfites is surveyed Sequence, methylation-specific polymerase chain reaction quantitatively and/or qualitatively, southern blotting technique method, restricted boundary mark genome scanning, At least one in the extension of mononucleotide primer, the island CpG microarray, joint sodium hydrogensulfite restriction endonuclease analysis and mass spectrum Kind.

Further, the reagent includes at least one set of Oligonucleolide primers pair in being selected from the group: SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 With SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22 and SEQ ID NO:23 and SEQ ID NO: 24。

Further, the reagent further includes the reagent for detecting miR-17-92 cluster expression quantity.

Further, the reagent of the detection miR-17-92 cluster expression quantity includes Oligonucleolide primers pair, it is preferable that institute State Oligonucleolide primers to sequence be selected from the group at least one set: SEQ ID NO:26 and SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.

Further, the reagent of the detection miR-17-92 cluster expression quantity includes reverse transcriptase primer and Taqman probe, excellent Selection of land, the sequence of the reverse transcriptase primer are SEQ ID NO:30 and/or SEQ ID NO:31, the sequence of the Taqman probe For SEQ ID NO:32 and/or SEQ ID NO:33.

Present invention firstly discovers that hypomethylation of the proto-oncogene miR-17-92 cluster in colorectal cancer is horizontal, it is straight with knot The occurrence and development of intestinal cancer are related.The methylation of miR-17-92 cluster can be used as the Biological indicators of lesion detection, and auxiliary diagnosis knot is straight Intestinal cancer improves and detects specificity, susceptibility, reduces error rate, to understand Tumorigenesis and diagnosis with highly important Clinical significance of MG.

The present invention uses the non-invasive detection methods of non-intrusion type, realizes the Accurate Diagnosis to colorectal cancer.The present invention is logical It crosses and extracts DNA from excrement or biopsy sample, after bisulf iotate-treated, carry out bisulfite sequencing and examine miR- 17-92 cluster methyl rate assesses the methylation in miR-17-92 cluster methylation sites (site CpG), to realize Colon and rectum The diagnosis of cancer；The limitation for overcoming the QPCR detection miRNA of mainstream, can be used as the Biological indicators of lesion detection, auxiliary is examined Disconnected colorectal cancer improves detection specificity, susceptibility, reduces error rate.

Detailed description of the invention

Fig. 1 is the site CpG in miR-17-92 cluster transcription initiation site upstream and downstream region；

Fig. 2 is the methylation of the miR-17-92 of colorectal cancer patients fecal specimens (T) and normal control fecal specimens (N) Rate；

Fig. 3 is the first of the miR-17-92 of colorectal cancer patients Colorectal Carcinoma sample (T) and cancer beside organism's sample (N) Base rate；

Fig. 4 is the ROC curve for detecting colorectal cancer patients fecal specimens and normal control fecal specimens；

Fig. 5 is the expression quantity of the miR-92a of colorectal cancer patients fecal specimens (T) and normal control fecal specimens (N)；

Fig. 6 is the expression quantity of the miR-17 of colorectal cancer patients fecal specimens (T) and normal control fecal specimens (N).

Specific embodiment

Present invention firstly discovers that original cancer non-coding RNA miR-17-92 cluster, that is, miR-17-92 cluster near transcriptional start sites Hypomethylation of the region in colorectal cancer is horizontal and related to the occurrence and development of colorectal cancer, therefore can be used as Colon and rectum Carcinoma marker.Research finds that the generation of miR-17-92 cluster and tumour is closely related.Researcher has found miR-92a in colon gland High expression in tumor and colon cancer.Moreover, miR-92a can directly inhibit anti-apoptosis factor BCL-2 to come in colon cancer tissue Mediating cell death, therefore can speculate that miR-92a plays a significant role in colorectal cancer occurrence and development.In addition, miR-17 Expression it is related to the prognosis of colorectal cancer patients.MiR-17-92 cluster member expresses significant raising, Ke Nengzuo in colorectal cancer For the new marker of diagnosis of colorectal carcinoma.At present in colorectal cancer, the research of miRNA methylation is less.MiR-17-92 cluster The effect to methylate in tumour does not have been reported that.The relationship of miR-17-92 cluster methylation level and tumour is inquired into, to understand tumour Pathogenesis and diagnosis have highly important Clinical significance of MG.

Current more and more evidences show that the expression of miRNA is by methylation modification regulation.And oneself has many research hairs Existing, miRNA is related with the occurrence and development of tumour.In colorectal cancer cell system, the island miR-137CpG high methylation.miR- 137 methylation levels are respectively 31.67% (± 17.25) and 33.09% (± 21.06) in colorectal cancer and adenoma sample, bright It is aobvious to be higher than normal tissue 7.70% (± 4.18) and cancer beside organism 10.27% (± 6.88).MiR-34 is in the cancer of the esophagus and normal right It is compared according to group, methyl rate increases.The methylation positive rate of colorectal cancer cancerous tissue miR34b/c gene is 95.2% (120/ 126), corresponding Carcinoma side normal tissue is 11.9% (15/126), and the two compares that there were significant differences (P < 0.01).MiRNA methyl The variation of change level is the important molecule feature of colorectal cancer, and detection miRNA methylation level is expected to as diagnosis of colorectal carcinoma One completely new tumor marker.

The morbidity and mortality of colorectal cancer are very high, and colorectal carcinoma is from benign development to pernicious, it usually needs 15 years More than, if can perform the operation after excision in early detection, cure rate is very high, up to 90% or more.How to select it is a kind of it is economical, The screening method convenient, compliance is good improves the recall rate of colorectal cancer, reduces the death rate, is that medical personnel is urgently to be solved Problem.Recent study persons search out a kind of convenient, noninvasive detection method, to cast-off cells in excrement, gene mutation, DNA methylation etc. is detected.Colon communicates with the outside world, and excrement is uniquely to be able to reflect a gastral window.Colon and rectum The cancer cell of cancer falls out or the dissociative DNA of secretion are excreted by excrement, and there is also the tumours of higher proportion in faeces DNA DNA.Since enteron aisle is alkaline environment, the DNA in excrement is not easy to be degraded by nucleic acid ribozyme.It is tied from excrement gene level Carcinoma of the rectum diagnosis, has non-invasive, randomness, convenience in terms of materials, is not necessarily to diet control, and faeces DNA methylates The foundation of analytical technology simplifies the method for sampling, reduces research expenditure.It has been reported that detection excrement miR34b/c gene methyl Change, it is possible to as diagnosis of colorectal carcinoma or the new method of screening.Colorectal cancer excrement miR34b/c methylation positive rate is 90.2% (111/123), is significantly higher than normal control 7.8% (5/64), and difference is statistically significant.Faeces DNA miR34b/c Sensibility for colorectal cancer early diagnosis is 90.2%, and specificity is 92.2%, as a result with acquire from same patient Colorectal cancer tissue result is close, prompts detection colorectal cancer patients excrement miR-96 gene methylation level in colorectal cancer There is major application value in diagnosis.

It has been found that miR-17-92 cluster member miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and MiR-92a-1 significantly high expression in colorectal cancer is likely to become the new marker of diagnosis of colorectal carcinoma, wherein miR-17-3p Susceptibility, specificity with miR-92 diagnosis colorectal cancer are respectively 64%, 70% and 89%, 70%.By analyzing miR-17- 92 cluster sequences find that there is the one section of longer island CpG sequence (being about 2kb) in its upstream, and CG content accounts for about 80%.And miR-17- 92 Cu CpG island sequences are highly conserved in the mammals such as people, mouse and rat, and miR-17-92 cluster is prompted to have by methyl A possibility that changing regulation and importance.Existing research finds the variation of miR-17-92 cluster methylation level and macrophage differentiation And pulmonary branches tracheae depauperation is related, but it does not have been reported that with cancer associated.Therefore have can for miR-17-92 methylation level It can be as new diagnosing tumor marker.

" methylation of miR-17-92 cluster " refers to that first occurs for the cluster near transcriptional start sites region miR-17-92 in the present invention Base, that is, miR-17-92 cluster transcription initiation site upstream of the present invention 3.5kb sequence is to the methyl of downstream 2kb sequence area Change.

Below in conjunction with attached drawing and specific embodiment, technical scheme is described further, but the present invention is not It is limited to these specific embodiments.Material used in embodiment, reagent etc., unless otherwise specified, commercially It arrives.

The screening of 1 methylation sites of embodiment (site CpG)

With California Santa Cruz (UCSC) university database analysis miR-17-92 cluster, bisulfites is designed (BSP) primer is sequenced, finds its site CpG, i.e. methylation sites.The site miR-17-92 cluster CpG concentrates on miR-17-92 cluster Transcription initiation site upstream 3.5kb sequence is between the 2kb sequence of downstream.Fig. 1 shows transcription initiation site upstream 2kb and downstream The site CpG between 2kb sequence, the present invention select miR-17-92 cluster transcription initiation site upstream 1.5kb sequence to downstream 1.5kb sequence (SED ID NO:25) is tested.

2 design of primers of embodiment

Design sequencing primer, that is, bisulfite sequencing (BSP) primer.Primer sequence is shown in Table 1.

1 methylation sites primer sequence of table

3 sample collection of embodiment

204 colorectal cancer patients excrement, Colorectal Carcinoma and cancer beside organism are collected, the operation of all cases cuts off mark This has all done routine pathology inspection, and preoperative to take tumor patient excrement to determine the diagnosis of cancer, sample is sent within half an hour DNA or RNA is extracted in laboratory, and the cDNA and DNA after RNA reverse transcription are saved in -20 DEG C；And the same period selects the age to match The control that 164 normal controls detect as excrement.All equal underwent operatives of patient treat and did not received radiotherapy in the preoperative Or chemotherapy.

Embodiment 4DNA is extracted

It is operated according to DNA extraction kit QIAmp DNA Mini Kit (article No. 51306).

1) 20 μ L Proteinase Ks are added in 1.5ml centrifuge tube；

2) 100-200mg fecal specimens are added；

3) it is added 400 μ L Sl Buffer jog 30 seconds and mixes；

4)60℃ 10min；

5) 12000rpm is centrifuged 5min, and supernatant is transferred in new centrifuge tube；

6) 400 μ L Binding Buffer are added；

7)60℃ 10min；

8) 100 μ L isopropanols are added, light mixing 5 seconds, centrifugation falls the liquid adhered on tube wall and lid in 10 seconds；

9) column is put into 2ml collecting pipe, transfer liquid is into column；

10) 8000rpm is centrifuged 1min, if liquid passes completely through column not yet after centrifugation, is centrifuged again until complete It passes through entirely；

11) column is transferred in new 2ml collecting pipe；

12) 500 μ L eluents 1 are added into column, 8000rpm is centrifuged 1min；

13) transfer column is into new 2ml collecting pipe；

14) 500 μ L eluents 2 are added into column, 8000rpm is centrifuged 1min；

15) 13000rpm is centrifuged 1min to completely remove ethyl alcohol；

16) 200 μ L E Buffer are added into 1.5ml collecting pipe in transfer column, stand 1 minute, make liquid abundant Diffusion；

17) 8000rpm is centrifuged 1min to elute.

Embodiment 5DNA carries out bisulf iotate-treated.

Referring to QIAGEN EptiTect Bisulfite Kit (article No. 59824) specification, the DNA of extracting is carried out sub- Disulfate processing.

(1) 85ul Bisulfite Mix, 35ul DNA Protect Buffer, 500ng are prepared in 0.2ml EP pipe DNA, moisturizing to 140ul system；

(2) in PCR instrument using following procedure react, 99 DEG C of denaturation 5min, 60 DEG C of incubation 25min, 99 DEG C of denaturation 5min, 60 DEG C of incubation 85min, 99 DEG C of denaturation 5min, 60 DEG C of incubation 175min；

(3) reaction terminates to take out 0.2ml EP pipe, and reaction solution is transferred in 1.5ml EP pipe；

(4) the BL buffer of 560 μ l Fresh, vortex oscillation is added；

(5) mixed liquor is added in centrifugal adsorbing column, puts on collecting pipe, maximum (top) speed centrifugation 1min, abandons liquid in collecting pipe Body；

(6) 500ul BW eluent is added in centrifugal adsorbing column, maximum (top) speed is centrifuged 1min, abandons liquid in collecting pipe；

(7) 500ul BD buffer is added in centrifugal adsorbing column, is stored at room temperature 15min；

(8) step (6) are repeated twice；

(9) maximum (top) speed is centrifuged 1min, abandons liquid in collecting pipe；

(10) centrifugal adsorbing column is placed in new collecting pipe, maximum (top) speed 1min；

(11) centrifugal adsorbing column is placed in clean 1.5ml EP to manage, 20ul deionized water is added into base for post matter, 12000rpm/min eluted dna, -20 DEG C of preservations.

Embodiment 6BSP amplification and detection

(1) BSP reacts

Reaction system is 25 μ l, reaction condition are as follows: 95 DEG C of initial denaturations 10min, 95 DEG C of denaturation 30s, 53.3 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 40 recycle.Products therefrom is determined using 2.0% agarose gel electrophoresis.

(2) carrier T connection and conversion

After purification by PCR product, it is attached by the specification of pMD18-T Vector, conversion to E.coli DH5 α.

(3) bacterium solution is sequenced

Ten clones are selected respectively and shake bacterium, are sequenced.

(4) sequencing result

If the site CpG methylates in former sequence, remained unchanged after bisulf iotate-treated, and individually C is equal It is converted into T.Methyl rate is the C that methylates ratio shared in all sites CG.Every group of practical first in each sample Base rate is added the practical methyl rate for being denoted as the sample.Compare the average methyl of colorectal cancer patients sample and normal human sample Rate, colorectal cancer patients sample interior according to its clinical pathology classification be compared respectively measurement data result with mean ± Standard deviation (x ± s) indicates.Using SPSS17.0 statistical software, 2 groups of means compare independent samples t test.Sequencing result such as table 2, 3 and Fig. 2, shown in 3.

The methyl rate of table 2 colorectal cancer patients fecal specimens and normal control fecal specimens

	Normal specimens methyl rate %	Colorectal cancer sample methyl rate %
			Primer pair 1	67.2±11.8	35.1±12.1
Primer pair 2	68.1±10.8	33.4±8.5
			Primer pair 3	62.2±9.4	32.3±9.3
Primer pair 4	72.2±11.3	34.3±10.4
			Primer pair 5	66.8±10.7	35.1±7.5
Primer pair 6	70.7±12.3	32.4±10.6
			Nested primer is to 1	64.0±9.4	28.9±10.5
Nested primer is to 2	64.3±8.5	28.6±8.7
			Nested primer is to 3	66.3±10.8	29.7±7.9

The methyl rate of table 3 Colorectal Carcinoma sample and cancer beside organism's sample

	Cancer beside organism sample methyl rate %	Colorectal cancer sample methyl rate %
			Primer pair 1	67.7±9.6	22.7±10.1
Primer pair 2	73.8±10.1	21.6±8.1
			Primer pair 3	63.0±8.5	23.6±9.6
Primer pair 4	74.7±8.3	21.4±5.4
			Primer pair 5	68.8±10.4	24.6±9.1
Primer pair 6	70.4±11.7	20.8±9.3
			Nested primer is to 1	64.1±9.9	24.5±7.5
Nested primer is to 2	66.5±9.3	21.1±6.7
			Nested primer is to 3	66.7±9.2	20.2±6.9

It can be seen that in fecal specimens from table 2 and Fig. 2, miR-17-92 cluster methylation sites are in colorectal cancer patients sample Middle hypomethylation, the hyper-methylation in normal reference sample.Compared with Normal group, miR-17-92 cluster methylation level (P < 0.001) is significantly reduced in colorectal cancer patients excrement.It can be seen that miR- in Colorectal Carcinoma from table 3 and Fig. 3 The methylation level of 17-92 cluster is significantly lower than cancer beside organism's sample.It is straight that prompt miR-17-92 cluster methylation level can be used as knot The mark of intestinal cancer diagnosing tumor.

Construct ROC (Receiver Operating Characteristic, ROC) tracing analysis, ROC curve such as Fig. 4 institute Show, to measure the AUC (area under the curve) compared in pairs about difference.Calculate youden index (Youden index)=true positives Rate-false positive rate=susceptibility-(1- specificity), taking the highest value of youden index is cutoff value (cut-off value).Thus obtain The cutoff value of miR-17-92 cluster methyl rate.Assess the methylation level area of the miR-17-92 cluster methylation sites of colorectal cancer Divide the normally diagnosis capability with the fecal sample of colorectal cancer patients.Under the optimal cutoff value of each pair of primer, detect quick Perception and specificity are as shown in table 4:

Diagnosis capability of 4 different primers of table to colorectal cancer

	Cutoff value (cutoff value)	Sensibility	Specificity
				Primer pair 1	48.10%	80.27	79.61
Primer pair 2	46.71%	83.49	77.12
				Primer pair 3	45.38%	84.71	75.16
Primer pair 4	44.91%	80.49	79.61
				Primer pair 5	46.74%	84.71	75.65
Primer pair 6	43.45%	83.88	77.61
				Nested primer is to 1	45.55%	84.1	79.12
Nested primer is to 2	42.11%	79.1	76.14
				Nested primer is to 3	47.65%	84.1	76.63

Table 4 the result shows that, miR-17-92 cluster DNA methylation assay colorectal cancer have relatively high sensitivity and specificity.

Variation of the embodiment 7miR-17-92 expression quantity in colorectal cancer patients

RNA extracting

The frost stool sample to have homogenized is clayed into power with liquid nitrogen grinding method, takes 100mg that 1mL Trizol is added, blows It beats vortex after mixing and vibrates 20s.The chloroform of about 1/5 volume is added, turns upside down and mixes well 1 minute or so, stand 5 at room temperature Minute.It 4 DEG C, carefully shifts supernatant after 12,000rpm centrifugations 15 minutes and enters new 1.5ml centrifuge tube, be added isometric different Propyl alcohol is gently mixed by inversion, and is stored at room temperature 10 minutes.4 DEG C, 12000rpm is centrifuged after ten minutes, is removed supernatant, is added into precipitating 70% ethyl alcohol of 2/5 volume, 4 DEG C, 12000rpm centrifuge washing precipitates 5 minutes.Supernatant is removed, is added after precipitating room temperature is dried suitable Water of the amount without RNA enzyme sufficiently dissolves.It is handled using the DNA enzymatic I of no RNA enzyme, QIAGEN RNeasy kits total serum IgE, in detail Thin operating principle and method are shown in kit specification.Total serum IgE quality is evaluated through agarose gel electrophoresis, obtains the 28S:18S of total serum IgE >=2, it can be preferable with preliminary judgement total serum IgE quality.

Q-PCR detection

The qPCR detection of miRNA uses the Trizol extracted total RNA of invitrogen, quantitative and quality inspection.Take 50-100ng Total serum IgE is template, uses the SuperScript III First- of reverse transcriptase primer and invitrogen for specific miRNA Strand Synthesis System kit synthesizes cDNA.Using cDNA as template, using for miR-17, miR-92a QPCR primer carries out quantitative detection to above-mentioned tumour-specific miRNAs using stem ring primer Taqman RT-PCR technology.In real time Quantitative fluorescent PCR is using small nuclear rna U6 as internal reference, target gene to be normalized, to ensure equal amount Compare the amount of target gene in sample.Using the relative quantification method in quantitative PCR, expression quantity is indicated with Δ Ct, Δ Ct= Ct_miR-92a-Ct_U6, the variation F=2 of expression quantity multiple^-ΔΔCtMethod calculates.Δ Δ Ct=(Ct in excrement_miR-92a-Ct_U6) tumour- (Ct_miR-92a-Ct_U6) normal control mean value.F indicate tumour patient excreta in target miRNAs expression relative to pairing just The often variation of control excreta, using P < 0.05 as statistically significant test stone.

QPCR primer:

MiR-92a:

Upstream primer: CCGTATTGCACTTGTCCCG (SEQ ID NO:26)

Downstream primer: GTGCAGGGTCCGAGGT (SEQ ID NO:27)

miR-17:

Upstream primer: CCGTATTGCACTTGTCCCG (SEQ ID NO:28)

Downstream primer: CAGCAAAGTGCTTACAGTGC (SEQ ID NO:29).

Reverse transcriptase primer:

Reverse transcription miR-92a:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGGC(SEQ ID NO:30)

Reverse transcription miR-17:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTACCT(SEQ ID NO:31)。

MiR-17Taqman probe: CTACCTGCACTATAAGCACTTTA (SEQ ID NO:32) miR-92a Taqman Probe: CAGGCCGGGACAAGTGCAATA (SEQ ID NO:33)

As a result such as Fig. 5, shown in Fig. 6.As can be seen that comparison healthy volunteer's fecal sample, the table of miR-17, miR-92a (P < 0.001) is obviously improved in colorectal cancer patients fecal sample up to amount.Using ROC curve method, (Receiver Operating Characteristics are bent Line) to research sample testing result analyze after, determine miR-17, miR-92a cutoff value.Its youden index maximum when institute Corresponding Cut-off value is respectively 31.25,30.75, its sensitivity is respectively 69.84%, 71.94% under the Ct value, specifically Property is respectively 71.67%, 77.25%.Joint-detection miR-17, miR-92a, under best Cut-off value, sensitivity is respectively 69.84%, 71.94%.It therefore, can be by the methylation water of miR-17-92 cluster in the diagnosis of colorectal cancer and risk assessment Gentle miR-17-92 cluster expression quantity as reference, improves detection specificity, susceptibility, reduces error rate simultaneously.

It will be understood by those skilled in the art that use of the invention is not only restricted to above-mentioned specific application.Just retouch herein State or the element-specific and/or feature described for, the present invention is also not limited to its preferred embodiment.It should be understood that The present invention is not limited to disclosed embodiment party's case or each embodiments, and illustrated by following following claims not departing from and Many is able to carry out in the case where the scope of the present invention of restriction to rearrange, modify and replace.

SEQUENCE LISTING

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<400> 8

ttctccaact actataatcc 20

<210> 9

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

gtatgagagg ggaggtttag gtatt 25

<210> 10

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

aaaaaacttt cctacccaaa aac 23

<210> 11

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

gagagtaggg tttttaggaa gtt 23

<210> 12

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

tccctttact acaatttaaa aacaaaaa 28

<210> 13

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

gtatgagagg ggaggtttag gtatt 25

<210> 14

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

ttaaaaacct actccaaacc aac 23

<210> 15

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

tttgaattat aatgtgaaga aggagg 26

<210> 16

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

cttcctaaaa accctactct cccta 25

<210> 17

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

gtaaagtaat aaattgtgat 20

<210> 18

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

cacacaacaa atttccctc 19

<210> 19

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

tagggagagt agggttttta ggaag 25

<210> 20

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

aacaaacccc actccctcat ta 22

<210> 21

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

gagggaaatt tgttgtgtg 19

<210> 22

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 22

cctcctttat atcccccaac cc 22

<210> 23

<211> 17

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 23

gggagggtgg gaggggg 17

<210> 24

<211> 15

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 24

cacactccca acaaa 15

<210> 25

<211> 2998

<212> DNA

<213> Homo sapiens

<400> 25

ttgaaacaga agataaagtt ttaaagaaaa aggcattgac atattagtca atgccgacca 60

aaagtaacgt aaaattagac ttgtaataaa attagcagtg ttttcctctt tgaaacgttc 120

ataaatgact atattacaga aatattagat attgtaaaat ttcagatttg gccttttatt 180

ttgatttaca aagaaaggaa tatctagaaa ctagactcaa taaaaaaaag agtagcaaaa 240

ctttaatcca ctatttttta tgtcatcact agtttgacaa ttggatttaa gcaaagcatg 300

ataaaataag tcaaagacag taaatgacaa tactgtcaag gtttgaatgt caaaaatttt 360

aacattgggg cattaaagac atcctttggt aatgtgtgtt ttacagttga cttgtacaat 420

cacgaaccag tgatatgtgc tttgcagtag ctacttacta gatttaaacc aatttataat 480

tgctgtattt ctcgtaaatg ttatgaatcc tcacttttaa caattatgtt ctgagaattc 540

cggaatttcc tgaaccacaa tgtgaagaag gaggtgctcc tgattgggct tcttttctca 600

gctttgtggt tgtttatgag gttctttccg agctatgcta tttctactgg agaacccaga 660

gttcttacta aatgcagctg ggcatgagag gggaggctca ggtactgcag ctgtgaaacc 720

tctgctcggg catgcgtttt ggtttacacg cggccaggtt aaggaaacac attgccacga 780

agtgtgcatt cacaagagtg tcgggtgtaa tgtatttatt tctgggtctc aaacgatctt 840

tgcagcgagt aattctgttt cgatgttaaa gtcactggat tttacttctc tggttccggt 900

tggcctggag caggccccta attacctcgg aaacccacca agggccggta aagtaataaa 960

ctgtgatcgc acagcgtaca cgtcgagtcc cagggagagc agggctccca ggaagccccg 1020

ggaagcgtgc gcttccagga gggtgtgcgg cgcgcggggg tctgcacgcg cgcagagctt 1080

gttaacggag ggcgtgccgg aggcgtggcg cgaggtgttc gggcgaggcg ctccgcgagg 1140

ctggcggcgg tgggcgggcc acgggggagc gcggcccggg gaaggctgcg cggcgccgcc 1200

agcggctccc ggctcccgct ctcccggccg ccaagaacga gccgccgtgg gcggggcctg 1260

cggtgattgg cgggcgggcg gggaggtcgg aagtactttg ttttttatgc taatgaggga 1320

gtggggcttg tccgtattta cgttgaggcg ggagccgccg cccttcattc acccacatgg 1380

tccttcgagg tgccgccgcc gccgcccgac ctgcgccttc gcgccacttc gcgccctcgg 1440

gcgaggccga gggggtgggg acgactaccg cagcgccgcc ggggctcgcg ctcctccgcg 1500

aagctctcct cgcggggcgg gccggccggc cgcacccccg gcctggggcc tccggtcgta 1560

gtaaagcgca ggcgggcggg gaggcgggag caggagcccg cggccggcca gccgaagatg 1620

gtggcggcta ctcctcctgg tgagtctgcc cgcccctccg gcgacggagg gaaacctgtt 1680

gtgtgcggcc cgggtctggc gggcggggcg gagcggcccg gggcggactg gcccggggca 1740

gcgtggcggc ggcggcgtgg ccggggcggg tctcggccgt tggccgcccc ggcgtgtggc 1800

agccgcatct ggctgccccc tcgctcgccc gcgggccggc ggaggggggc agggccgggg 1860

cgggagggtg ggagggggcg gcgtgcgcgt ggcggccgcg cccgggaccc gcgcagaccc 1920

tgcctgggcc gacccgaagg cgggtgggcg gacggcgaac acaatggccc ctcggggaga 1980

ggacgtgcga ggcccgtgcc ttctccgggg cccggggcgc gcgcggggcg tggggtctct 2040

gggtaggaaa gtttctcccg agggcgagag ttaaagcgcc tccagaacaa agcggcggcg 2100

gcggcggcac atggggcagg ccgcgggccg ggagggggcg cgcccacgag gtacctgcgc 2160

gccagcgggc ggcgtggcgt gggcgggagc ccgcggttcc ccaaactttg tacgcgcgag 2220

ggtgggcgga ggggcgccga gatcggcgcg gcctgggcgc cacccccgct ccgcgtgggc 2280

tttgttagcc cgcgtgggca gcctcggggc ggggcccgca acttccccgc cgtggccctc 2340

ggaggaggcc gcagtcggcc tcagccgcgg cgtggagccg cctgcgcccg gccgcttgct 2400

gggagtgtgg cgcgggaggg ccagcccggc tcggcgggag cggcgtcccc gccgccatgt 2460

tcctgcgggg cgggctgcac gggggtgagg gcgggggaca tggcggcgac tgcgcgccgc 2520

cgccgattgt tcccggctta ggcctcgggc cgcgtgcgac gggcaccgcg gccgcggagc 2580

ccccgcccct ctgggccggg ctcggggggg ctgggggaca caaaggaggg gcggcgcgcc 2640

cgcgtccccg ccgcactcgg gcctcggcgc cgccggtcgc cgcgcggctg ccgccgggaa 2700

acgggttggg ggggttgccg cgtccggcgg ggcctgactc tgacccgccg ccccctggcg 2760

gctacgcgga gaatcgcagg gccgcgctcc cccttgtgcg acatgtgctg ccggcccggg 2820

ctccatgagc gtggcgggca ctttgcagtc tcgggtgttc ctgcccggtc ttctgttcct 2880

aaactgcagc aaagggaaaa ggaactgaaa aaggcaggct cgtcgttgca atatcaccaa 2940

aagagaaaat taacggcatg ccatcaggac cacagcagtt ggagaaacaa ctctttat 2998

<210> 26

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 26

ccgtattgca cttgtcccg 19

<210> 27

<211> 16

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 27

gtgcagggtc cgaggt 16

<210> 28

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 28

ccgtattgca cttgtcccg 19

<210> 29

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 29

cagcaaagtg cttacagtgc 20

<210> 30

<211> 50

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 30

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacacaggc 50

<210> 31

<211> 50

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 31

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacctacct 50

<210> 32

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 32

ctacctgcac tataagcact tta 23

<210> 33

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 33

caggccggga caagtgcaat a 21

Claims

1. purposes of the reagent of the sequence methylation level to be measured in sample to be tested in preparation diagnosis of colorectal carcinoma reagent is detected, It is characterized in that, the sequence to be measured is located at miR-17-92 cluster transcription initiation site upstream 3.5kb sequence to downstream 2kb sequence Between.

2. purposes according to claim 1, which is characterized in that the diagnosis of colorectal carcinoma reagent is for carrying out colorectal cancer Diagnosis, Risk of Colorectal Cancer assessment and/or treatment of colorectal cancer recruitment evaluation.

3. purposes according to claim 1, which is characterized in that the miR-17-92 cluster is located at human chromosomal 13q31.3 On, GI:387942364.

4. purposes according to claim 1, which is characterized in that the sequence to be measured is located at miR-17-92 cluster transcription initiation Site upstream 1.5kb sequence is between the 1.5kb sequence of downstream, it is preferable that the sequence to be measured is SEQ ID NO:25 or SEQ The reverse complementary sequence of ID NO:25.

5. purposes according to claim 1, which is characterized in that the sample to be tested is selected from excrement, blood and biopsy At least one of.

6. purposes according to claim 1, which is characterized in that the method for the detection sequence methylation level to be measured is selected from Pyrosequencing, bisulfite sequencing, methylation-specific polymerase chain reaction quantitatively and/or qualitatively, southern blotting technique Method, restricted boundary mark genome scanning, the extension of mononucleotide primer, the island CpG microarray, joint sodium hydrogensulfite restriction enzyme At least one of enzyme assay and mass spectrum.

7. purposes according to claim 1, which is characterized in that the reagent includes the few core of at least one set in being selected from the group Thuja acid primer pair: SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22 and SEQ ID NO:23 and SEQ ID NO:24.

8. purposes according to any one of claims 1 to 7, which is characterized in that the reagent further includes detection miR-17- The reagent of 92 cluster expression quantity.

9. purposes according to claim 8, which is characterized in that it is described detection miR-17-92 cluster expression quantity reagent include Oligonucleolide primers pair, it is preferable that the Oligonucleolide primers to sequence be selected from the group at least one set: SEQ ID NO:26 With SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.

10. purposes according to claim 8, which is characterized in that it is described detection miR-17-92 cluster expression quantity reagent include Reverse transcriptase primer and Taqman probe, it is preferable that the sequence of the reverse transcriptase primer is SEQ ID NO:30 and/or SEQ ID NO:31, the sequence of the Taqman probe are SEQ ID NO:32 and/or SEQ ID NO:33.