CN103320504A - Detection of microRNAs in excreta as early diagnosis biomarker of lung cancer, colorectal cancer and bladder cancer - Google Patents
Detection of microRNAs in excreta as early diagnosis biomarker of lung cancer, colorectal cancer and bladder cancer Download PDFInfo
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Abstract
The invention discloses a method for detection of microRNAs in excreta (sputum, faeces and urine) as a biomarker for early screening and diagnosing lung cancer, colorectal cancer and bladder cancer. The method comprises: (1) screening abnormal expression microRNAs existed in tissues of the lung cancer, the colorectal cancer and the bladder cancer; (2) by using resection tumor samples from patients of the lung cancer, the colorectal cancer and the bladder cancer, verifying the screened microRNAs in the tumor tissues; and (3) detecting expressions of the microRNAs in samples of sputum, faeces and urine from the patients of the lung cancer, the colorectal cancer and the bladder cancer, analyzing correlation with expression in the tissues, and confirming the microRNAs for early screening and diagnosis. The method is substantially characterized in that the microRNAs in the excreta is taken as the biomarker for early screening and diagnosing the cancers, and the method helps to realize noninvasive screening for the cancer patients and possesses a wide application scope.
Description
Technical field
The invention belongs to the medical diagnostic techniqu field, be specifically related to the screening of tumour-specific microRNAs, for foundation and the optimization of the detection system of tumour-specific microRNAs, and the application in screening for cancer and early diagnosis.
Background technology
The tissue of epithelial cell canceration comprises esophagus cancer, cancer of the stomach, Pancreatic neoplasms, the colorectal cancer of Digestive tract, the lung cancer of respiratory system and the bladder cancer of urinary system etc.The mortality ratio of these cancers all is year by year ascendant trend, and can effectively reduce mortality ratio to high risk population's examination and early diagnosis, but does not have now desirable nothing wound screening method.MicroRNAs (miRNAs) is the non-coding microRNA of performance wide application in gene expression regulation, in swollen neoplastic process unconventionality expression is often arranged, and special miRNAs express spectra can be distinguished dissimilar cancer.Behind the epithelial cell canceration, tumour cell can come off, and excrete with movement, so the miRNAs in the movement (comprising ight soil, urine, sputum) can be used as a kind of nothing wound tumor markers of being potential very much.
Aspect ight soil miRNAs diagnosis digestive system tumor, 2009, show in the research of Ahmed et al to the colorectal cancer examination, there be the abnormal expression of 7 kinds of miRNAs in Patients with Colorectal Cancer cancerous issue and ight soil to increase (miR-21, miR-106a, miR-96, miR-203, miR-20a, miR-326 and miR-92), there are in addition 7 kinds to express the (miR-320 that descends, miR-126, miR-484-5p, miR-143, miR-145, miR-16 and miR-125b) (Ahmed FE, Jeffries CD, Vos PW, et al.Diagnostic microRNA markers for screening sporadic human colon cancer and active ulcerative colitis in stool and tissue.Cancer Genomics Proteomics, 2009,6 (5): 281-295).2010, Link et al finds the many of miR-21 and the expression ratio colorectal cancer patients of miR-106a in knot adenomas patient ight soil, and its expression amount can be with TNM increasing and reduce (LinkA by stages, Balaguer F, Shen Y, et al.Fecal MicroRNAs novel biomarkers for colon cancer screening.Cancer Epidemiol Biomarkers Prev, 2010,19 (7): 1766-1774).2012, Wu et al thinks that miR-92a is higher than respectively cancer beside organism and normal people's ight soil at cancerous issue and the content in the ight soil of Patients with Colorectal Cancer, and compare with normal people's ight soil, the expression amount of miR-92a in polyp of colon patient ight soil also obviously increases, show by further data analysis, the miR-92a diagnosis distal colorectal rectum cancer is higher than the sensitivity of near-end colorectal cancer, diagnose high-risk adenoma than high (the Wu CW of the sensitivity of polyp of colon, Ng SS, Dong YJ, et al.Detection of miR-92a and miR-21 in stool samples as potential screening biomarkers for colorectal cancer and polyps.Gut, 2012,61 (5): 739-745).And between 2010-2012, there are successively some study group to confirm, miR-144, miR-17-92 bunch and miR-135 obviously increase in colorectal cancer patients ight soil, the sensitivity of both Colorectal Cancer Diagnosis and specificity are respectively 74.1% and 79.0% (Li JM afterwards, Zhao RH, Li ST, et al.Down-regulation of fecal miR-143and miR-145as potential markers for colorectal cancer.Saudi Med J, 2012,33 (1): 24-29.Koga Y, Yasunaga M, Takahashi A, et al.MicroRNA expression profiling of exfoliated colonocytes isolated from feces for colorectal cancer screening.Cancer Prev Res (Phila), 2010,3 (11): 1435-1442.Kalimutho M, Del Vecchio Blanco G, Di Cecilia S, et al.Differential expression of miR-144*as a novel fecal-based diagnostic marker for colorectal cancer.J Gastroenterol, 2011,46 (12): 1391-1402.).
2012, Link et al finds that the expression amount of 4 kinds of miRNAs (miR-216a, miR-196a, miR-143 and miR-155) in the ight soil is high expression level at Normal group, Patients With Chronic Pancreatitis takes second place, Pancreas cancer patients is low (the Link A of expression, Becker V, Goel A, et al.Feasibility of Fecal MicroRNAs Novel Biomarkers for Pancreatic Cancer.PLoS One, 2012,7 (8): e42933.).That Ren et al then thinks the most effective differentiating pancreatic cancer is miR-181b and miR-210 (Ren Y, Gao J, Liu JQ, et al.Differential signature of fecal microRNAs in patients with pancreatic cancer. Mol Med Report, 2012,6 (1): 201-209).
In urine aspect the miRNAs diagnosing bladder cancer, 2011, Yamada et al finds bladder transitional cell carcinoma (Urothelial Carcinoma, UC) expression amount of miR-96 and miR-183 increases unusually in the urine, the two sensitivity and specificity that is used for differentiation UC and Healthy People is respectively 71% and 89.2%, 74% and 77.3%, and the patient UC miR-96 of 61.4% urine exfoliative cytology inspection feminine gender is positive, therefore Urine exfoliated cells is learned and checked and the join together susceptibility of examination UC of the detection of miR-96 can rise to 78.2% (Yamada Y, Enokida H, Kojima S, et al.MiR-96and miR-183detection in urine serve as potential tumor markers of urothelial carcinoma:correlation with stage and grade, and comparison with urinary cytology.Cancer Sci, 2011,102 (3): 522-529).2012, Snowdon et al finds that the sensitivity that miR-125b and miR-126 in the transitional cell bladder carcinoma unite diagnosis reaches 80%, specificity is 100% (Snowdon J, Boag S, Feilotter H, et al.A pilot study of urinary microRNA as a biomarker for urothelial cancer.Can Urol Assoc J, 2012,15:1-5), and Miahet al proof miR-135b, the sensitivity of miR-15b and miR-1224-3p joint-detection bladder cancer can reach 94.1% (Miah S, Dudziec E, Drayton RM, et al.An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer.Br J Cancer, 2012,107 (1): 123-128.).MiR-452 (AUC=0.848) and miR-222 (AUC=0.718) diagnosing bladder cancer also have very high accuracy rate (Puerta-Gil P in the Puerta-Gil et al research discovery urine in addition, Garc í a-Baquero R, Jia AY, et al.miR-143, miR-222, and miR-452are useful as tumor stratification and noninvasive diagnostic biomarkers for bladder cancer.Am J Pathol, 2012,180 (5): 1808-1815).
2012, Wang et al thinks that miRNAs has different meanings in these two parts of arena and centrifugal rear supernatant liquor in the urine, they are studied two portions, discovery is compared with control group, miR-200 family in the bladder cancer patients arena (miR-200a, miR-200b, miR-200c, miR-141 and miR-429), miR-192 and miR-155 express and reduce, and miR-205 and miR-146 be indifference then; In supernatant liquor, miR-192 expresses also reduction but the miR-155 expression is increased, other several miRNAs then with control group indifference (Wang G, Chan ES, Kwan BC, et al.Expression of microRNAs in the Urine of Patients With Bladder Cancer.Clin Genitourin Cancer, 2012,10 (2): 106-113).Free two kinds of miRNAs (miR-145 and miR-200a) also are proved and can be used as the mark that bladder cancer is made a definite diagnosis and recurred in the Yun et al confirmation urine in addition, wherein miR-145 is used for diagnosing non-flesh layer invasive bladder cancer (Non Muscle Invasive Bladder Cancer, NMIBC) and flesh layer invasive bladder cancer (Muscle Invasive Bladder Cancer, MIBC) sensitivity is respectively 77.8% and 84.1%, specificity all is 61.1%, and its expression level is relevant with classification; MiR-200a then can be used as the independentpredictor of NMIBC recurrence, its low expression is often indicating high relapse rate (Yun SJ, JeongP, Kim WT, et al.Cell-free microRNAs in urine as diagnostic and prognostic biomarkers of bladder cancer.Int J Oncol, 2012,41 (5): 1871-1878).
Aspect sputum miRNAs diagnosing, 2010, Xie et al
[31]Find that miR-21 is at nonsmall-cell lung cancer (Non Small Cell Lung Cancer, NSCLC) content in patient's sputum increases than the normal people is obvious, miR-21 all is high expression level in the sputum of phlegm cytology checking positive patient, have that miR-21 is high expression level in half phlegm cytology checking negative patient's the sputum, and miR-21 just has higher level (Xie Y in the lung cancer in early days, Todd NW, Liu Z, et al.Altered miRNA expression in sputum for diagnosis of non-small cell lung cancer.Lung Cancer, 2010,67 (2): 170-176.).Yu et al subsequently
[32]Tissue sample and the healthy tissues sample of getting adenocarcinoma of lung compare, discovery has 7 kinds of miRNAs to have notable difference between the two, wherein 4 kinds (miR-21, miR-182, miR-375 and miR-200b) expresses rising in cancerous tissue, 3 kinds (miR-486, miR-126 and miR-145) expresses reduction in cancerous tissue.Then analyze the content of these 7 kinds of miRNAs in 36 routine I phase patients with lung adenocarcinomas and control group sputum, find their variations in sputum and the tissue in consistent.By the analysis of data being found wherein the value of miR-21, miR-486, miR-375, miR-200b associating Diagnosis of pulmonary gland cancer is the highest, sensitivity and specificity are respectively 80.6% and 91.7%.Selecting one group of independent sample else studies these 4 kinds of miRNAs again, find that their diagnostic values in different tumor tissues types are different, sensitivity and specificity that they unite diagnosis are 80.6% and 92.5% in gland cancer, are 64.1% and 71.3% in the squama cancer; Peripheral type carcinoma of lung is 78.3% and 93.8%, central type carcinoma of lung is 65.8% and 70.9%, no significant difference (Yu L then in I, II, III, IV phase lung cancer but, Todd NW, Xing L, et al.Early detection of lung adenocarcinoma in sputum by a panel of microRNA markers.Int J Cancer, 2010,127 (12): 2870-2878.).Consistent with above-mentioned experimental technique and step, Xing et al
[33]Confirm miR-205 in the lung squamous cell carcinoma cancers, miR-210 and miR-708 express and increase, miR-126, miR-139 and miR-429 express and reduce, miR-205 wherein, the associating diagnostic value of miR-210 and miR-708 is the highest, its sensitivity and specificity are respectively 73% and 96%, their expression amounts in I phase lung squamous cancer are just very high, and its expression level does not change (Xing L with the progress of lung cancer, Todd NW, Yu L, et al.Early detection of squamous cell lung cancer in sputum by a panel of microRNA markers.Mod Pathol, 2010,23 (8): 1157-1164.).
2012, Roa et al
[34]Detect sputum miRNAs Optimum combinational scheme (miR-21 Deng utilizing RT-PCR to detect to add the cluster analysis method to screen one, miR-143, miR-155, miR-210, miR-372), the sensitivity of early diagnosis NSCLC and specificity reach 83.3% and 100% (Roa WH, Kim JO, Razzak R, et al.Sputum MicroRNA Profiling:A Novel Approach for the Early Detection of Non-Small Cell Lung Cancer.Clin Invest Med, 2012,35 (5): E271.).
Open book (the publication number: US2011/0086353 of application for a patent for invention; Country origin: the U.S.; Open day: on April 14th, 2011) disclose the mark that diagnosis, prognosis and treatment are relevant that comprises that the unconventionality expression of miRNAs (miR-21, miR-106a etc.) in the ight soil can be used as digestive system tumor/colorectal cancer.And invented a kind of detection method that does not need to extract in advance miRNA.
Open book (the publication number: US2011/0136124 of application for a patent for invention; Country origin: the U.S.; Open day: on June 9th, 2011) disclose miRNAs (miR-21 in the sputum, miR-155, miR-210, miR-143, miR-372) with the contacting of lung cancer, comprise and differentiate lung cancer and other cancers, distinguish different lung cancer cell lines and the differential expression between the Healthy People of lung cancer therapy front and back, the Healthy People that smoking history is arranged and non-smoking thereof.
Summary of the invention:
Tumor markers and other noninvasive examination means exist sensitivity low at present, and the problem of poor specificity in order to address this problem, the objective of the invention is to choose the miRNAs of tumour-specific as new tumor markers; Another object of the present invention is to set up a kind of simple, quick, economic miRNAs detection system; A further object of the invention just combines the tumour miRNAs highly sensitive, that specificity is good and this easily detection system sets up a kind of test kit with commercial value for examination and early diagnosis tumour.
The object of the present invention is achieved like this, namely choose miRNAs with Tumor-assaciated by the report of the past document, and confirm in tumor tissues, to exist their unconventionality expression to compose, therefrom simplify out several miRNAss the most relevant with early diagnosis of tumor, in conjunction with the RT-RCR technology, obtain the early diagnosis of tumor test kit highly sensitive, that specificity is good after the optimization.The present invention includes the content of three aspects:: screening tumour-specific miRNAs, foundation detect tumour miRNAs detection system and the application in early diagnosis of tumor thereof, its innovative point is to utilize coming off of primary tumor tumour cell, may become early diagnosis of tumor by slop detection tumour-specific miRNAs and newly indicate; Utilize the RT-PCR technology, develop a kind of have highly sensitive, specificity is high, flux is high and the movement tumour-specific miRNAs immue quantitative detection reagent box of low cost and other advantages, and this test kit for early diagnosis of tumor, instruct individualized treatment and early discovery tumor recurrence to shift and judge that the prognosis of tumour provides a kind of new reliability index.
Concrete technical scheme is as follows:
1. the screening of tumour-specific miRNAs.Described tumour miRNAs is that the early diagnosis degree of correlation with tumour that is confirmed by the past document is high, selected among the report miRNAs often.
Lung cancer: miR-21, miR-155, miR-205, miR-125b, miR-708, miR-210, miR-200b, miR-106a, miR-126, miR-486-5p
Colorectal cancer: miR-21, miR-106a, miR-96, miR-326, miR-92, miR-126, miR-484-5p, miR-143, miR-145
Bladder cancer: miR-200a, miR-96, miR-183, miR-126, miR-135b, miR-182, miR-125b, miR-15b, miR-1224-3p, miR-145
The articles of reference relevant with screening lung cancer miRNAs, be Boeri M, Verri C, Conte D, et al.MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer.Proc Natl Acad Sci USA, 2011,108 (9): 3713-3718; Guan P, Yin Z, Li X, et al.Meta-analysis of human lung cancer microRNA expression profiling studies comparing cancer tissues with normal tissues[J] .J Exp Clin Cancer Res, 2012,31 (1): 54.
The articles of reference relevant with screening colorectal cancer miRNAs, be Ahmed FE, Jeffries CD, Vos PW, et al.Diagnostic microRNA markers for screening sporadic human colon cancer and active ulcerative colitis in stool and tissue.Cancer Genomics Proteomics, 2009,6 (5): 281-295.
The articles of reference relevant with screening bladder cancer miRNAs, be Wang G, Chan ES, Kwan BC, et al.Expression of microRNAs in the Urine of Patients With Bladder Cancer.Clin Genitourin Cancer, 2012,10 (2): 106-113; Snowdon J, Boag S, Feilotter H, et al.A pilot study of urinary microRNA as a biomarker for urothelial cancer.Can Urol Assoc J, 2012,15:1-5.; Miah S, Dudziec E, Drayton RM, et al.An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer.Br J Cancer, 2012,107 (1): 123-128.; Yun SJ, Jeong P, Kim WT, et al.Cell-free microRNAs in urine as diagnostic and prognostic biomarkers of bladder cancer.Int J Oncol, 2012,41 (5): 1871-1878.
2. the checking of described tumour-specific miRNAs.Concrete steps can be as follows: excision cancerous tissue and the cancer beside organism of collecting respectively 16 routine patients with lung cancer, 15 routine colorectal cancer patients and 12 bladder cancer patients that clinical pathology makes a definite diagnosis.After extracting total RNA, utilize Taqman human miRNA array A that the difference that exists in above-mentioned tumour-specific miRNAs cancerous tissue and the non-cancer tissue is verified.
1. confirm to compare with the cancer beside organism of matching, miR-21, miR-155, miR-205, miR-125b, miR-708, miR-210, miR-200b, miR-106a express significantly increase in the cancerous lung tissue, and miR-126, miR-486-5p express significantly and reduce.
2. confirm to compare with the cancer beside organism of matching, miR-21, miR-106a, miR-96, miR-326, miR-92 express significantly increase in the Colorectal Carcinoma, and miR-126, miR-484-5p, miR-143, miR-145 express significantly and reduce
3. confirm to compare with the cancer beside organism of matching, miR-200a, miR-96, miR-183, miR-126, miR-135b, miR-182 express significantly increase in the Bladder Cancer, and miR-125b, miR-15b, miR-1224-3p, miR-145 express significantly and reduce.
3. the expression of described tumour-specific miRNAs in movement.Concrete steps can be as follows: collect respectively sputum, ight soil and the urine of 48 routine patients with lung cancer, 49 routine colorectal cancer patients and 52 routine bladder cancer patients that clinical pathology makes a definite diagnosis and 48 routine healthy volunteers' sputum, ight soil and urine.After extracting total RNA, utilize stem ring primer Taqman RT-PCR technology that above-mentioned tumour-specific miRNAs is carried out detection by quantitative.Real-time fluorescence quantitative PCR, comes target gene is carried out normalized as internal reference with small nuclear rna U6, with the amount of comparison object gene in the sample of guaranteeing equal amount.Adopt the relative quantification method in the quantitative PCR, expression amount represents with Δ Ct, Δ Ct=Ct
MiRNA-Ct
U6, the variation F=2 of expression amount multiple
-Δ Δ CtMethod is calculated.Δ Δ Ct=(Ct in the movement
MiRNA-Ct
U6) tumour-(Ct
MiRNA-Ct
U6) the normal control average.The expression that F represents target miRNAs in the tumour patient movement is with respect to the excremental variation of normal control of pairing, with P＜0.05 as the inspecting standard that statistical significance is arranged.And determine that these miRNAs are used for specificity and the sensitivity of examination and early diagnosis tumour.
Description of drawings:
Fig. 1 is the differential expression of miRNAs in cancerous lung tissue and cancer beside organism figure as a result among the embodiment two.
Fig. 2 is the differential expression of miRNAs in colorectal carcinoma and cancer beside organism figure as a result among the embodiment two.
Fig. 3 is the differential expression of miRNAs in Bladder Cancer and cancer beside organism figure as a result among the embodiment two.
Specific embodiments
One, excremental collection and processing
Embodiment 1:
The tissue sample collection places places liquid nitrogen container in the cryopreservation tube, transfer to-80 ℃ of Refrigerator stores.Be stored in 4 ℃ after the sputum specimen collection, and within a week, process.Every portion of sputum is divided in the RNase free Eppendorf tube that installs to 1.5ml by 200 μ l, and add 400 μ l Sputolysin
TM(0.1mg/ml, sigma) solution, whirlpool mixing are stored in the sputum that homogenizes-20 ℃ at last until after the dissolving of the sputum of stickiness, hatched 30 minutes for 37 ℃.After stool sample is collected, divide by every part of ight soil 200mg to install in the cryopreservation tube, be stored in-80 ℃.Urine sample collecting is centrifugal 10 minutes of 50ml approximately, and the precipitation that obtains after supernatant is removed washes twice with PBS, and is frozen in-80 ℃.Within a week, extract RNA after all samples are frozen.
Two, the extraction of RNA
Embodiment 2:
With 1000 μ l Trizol
TM(invitrogen) join in the centrifuge tube of storage of specimens (comprise implement the 25mg frozen tissue sample described in sharp 1 with the sputum that the liquid nitrogen grinding method is clayed into power, 200 μ l have homogenized, freezing stool sample with the liquid nitrogen grinding method clay into power, the precipitation of 50ml urine after centrifugal), blew and beat behind the mixing vortex oscillation 20 seconds.Then adding 20 seconds room temperatures of 200 μ l chloroform whirlpool mixings placed 5 minutes.4 ℃ in mixture was separated into water and red organic phase in centrifugal 15 minutes.Water (approximately 500 μ l) carefully is transferred in the clean 1.5ml centrifuge tube, adds glycogen coprecipitator (Ambion) and the 5000 μ l Virahols of 4 μ l, room temperature was placed 20 minutes behind the soft mixing.Then 12000rpm is centrifugal 10 minutes, and 4 ℃ precipitated RNA in centrifugal 15 minutes, 4 ℃ throw out 75% washing with alcohol, centrifugal 2 minutes of 8000rpm repeats twice.Then make RNA be dissolved in-20 ℃ of preservations in the 105 μ l RNase free water.For concentration and the content that detects RNA, the RNA of 5 μ l is diluted in the RNase free water of 95 μ l, use Beckman DU
TM7500 spectrophotometer measurements.The content (μ g) of RNA in the 200 μ l samples should be between 0.5 to 3.0 μ g.A
260And A
280Ratio should be greater than 1.
RNA(μg/μl)＝OD
260×20×40
RNA(μg)＝OD
260×20×40×0.1
Three, the detection by quantitative of miRNAs
Embodiment 3:
The reverse transcription of miRNA uses
MicroRNA Reverse Transcription Kit, primer and StepOnePlus
TMReal-time quantitative PCR system (Applied Biosystems Inc.) carries out to specifications.The RT Master Mix according to the form below configuration of each reaction:
RT Master Mix is placed the PCR pipe of Nuclease-free, softly mix and be placed on ice.The RNA that extracts among 3.0 μ lmiRNA primers and the 5 μ l embodiment 2 is added among every part of RT Master Mix, and the final reaction system is 15 μ l, and 1500rpm is centrifugal two minutes behind the mixing.16 ℃ of 30min on the PCR instrument, 42 ℃ of 40min, 85 ℃ of 5min placed the product of generation-70 ℃ preservation approximately 50 minutes for 4 ℃.
Embodiment 4:
Use
Universal PCR Master Mix,
MicroRNA Assay, primer, probe and StepOnePlus
TMReal-time quantitative PCR system (Applied Biosystems Inc.) also carries out the qRT-PCR detection by quantitative to target miRNA to specifications.
Obtaining with Ct value form of real-time fluorescence quantitative PCR visual result, all samples increase to target miRNA and confidential reference items U6 simultaneously, each sample triplicate.The PCR reaction totally is 20 μ l.
Mixture softly mixes and places on ice, and then 1500rpm is centrifugal 2 minutes.95 ℃ of sex change are 10 minutes on the PCR instrument, with 95 ℃ 15 seconds, 60 ℃ 1 minute, carry out 40 circulations.
Table 1 and lung cancer, colorectal cancer, the miRNAs that the bladder cancer early diagnosis is relevant
The expression amount of table 2 lung cancer specificity miRNAs in patients with lung cancer and healthy volunteer's sputum (the miRNA expression amount represents with average and the standard deviation of Δ Ct, take U6 as confidential reference items, and P＜0.001)
The expression amount of table 3 colorectal cancer specificity miRNAs in colorectal cancer patients and healthy volunteer's ight soil (the miRNA expression amount represents with average and the standard deviation of Δ Ct, take U6 as confidential reference items, and P＜0.001)
The expression amount of table 4 bladder cancer specificity miRNAs in bladder cancer patients and healthy volunteer's urine (the miRNA expression amount represents with average and the standard deviation of Δ Ct, take U6 as confidential reference items, and P＜0.001)
The present invention can summarize with other the specific form without prejudice to spirit of the present invention or principal character.Therefore, no matter from which point, above-mentioned embodiment of the present invention all can only be thought can not limit the present invention to explanation of the present invention, claims have been pointed out scope of the present invention, therefore, in the implication suitable with claims of the present invention and any change in the scope, all should think to comprise within the scope of the present invention.
Claims (9)
- One kind be used for cancer high risk population examination and the method for early diagnosis, comprise following step:1. collect crowd's to be checked movement sample (sputum, ight soil, urine).2. detect the expression of tumour-specific miRNAs in the movement sample.3. determine whether crowd to be checked is the high risk population who suffers from cancer (lung cancer, colorectal cancer, bladder cancer), its excremental miRNAs express spectra must contain a miRNAs unconventionality expression spectrum of having determined and two or more at least other tumour-specific miRNAs abnormal expression.
- 2. according to right 1 described cancer, it is characterized in that comprising lung cancer, colorectal cancer and bladder cancer, described lung cancer, colorectal cancer and bladder cancer also comprise the secondary tumors that is transferred to its hetero-organizations such as brain, mammary gland, prostate gland by carcinoma in situ.
- 3. according to right 1 described tumour-specific miRNAs, it is characterized in that the miRNAs sequence and the hair clip precursor sequence thereof that comprise that it is ripe.
- 4. according to right 1 described tumour-specific miRNAs and definite miRNAs unconventionality expression spectrum, it is characterized in that being drawn by statistical analysis.
- 5. according to right 3 described statistical analysis, it is characterized in that comprising the hierarchial-cluster analysis method.
- 6. according to right 1 described tumour-specific miRNAs, it is characterized in that comprising:1. lung cancer: miR-21, miR-155, miR-205, miR-125b, miR-708, miR-210, miR-200b, miR-106a, miR-126, miR-486-5p2. colorectal cancer: miR-21, miR-106a, miR-96, miR-326, miR-92, miR-126, miR-484-5p, miR-143, miR-1453. bladder cancer: miR-200a, miR-96, miR-183, miR-126, miR-135b, miR-182, miR-125b, miR-15b, miR-1224-3p, miR-145.
- 7. according to the right 1 described miRNAs unconventionality expression spectrum of having determined, it is characterized in that comprising:1. lung cancer: miR-21, miR-155, miR-205, miR-125b2. colorectal cancer: miR-484-5p, miR-145, miR-21, miR-106a3. bladder cancer: miR-1224-3p, miR-15b, miR-200a, miR-96.
- 8. should contain at least two or more other tumour-specific miRNAs according to right 1 is described, it is characterized in that comprising:1. lung cancer: miR-708, miR-210, miR-200b, miR-106a, miR-126, miR-486-5p2. colorectal cancer: miR-96, miR-326, miR-92, miR-126,, miR-1433. bladder cancer: miR-183, miR-126, miR-135b, miR-182, miR-125b, miR-145.
- 9. according to the expression of right 1 described miRNAs, it is characterized in that using the RT-qPCR method to detect.
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