CN103627705A - PiRNA biomarker related to bladder cancer and application thereof - Google Patents

PiRNA biomarker related to bladder cancer and application thereof Download PDF

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CN103627705A
CN103627705A CN201310516529.4A CN201310516529A CN103627705A CN 103627705 A CN103627705 A CN 103627705A CN 201310516529 A CN201310516529 A CN 201310516529A CN 103627705 A CN103627705 A CN 103627705A
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dna
artificial sequence
pirna
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CN103627705B (en
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张正东
王美林
储海燕
殷长军
仝娜
袁琳
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Nanjing University
Nanjing Medical University
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Abstract

The invention discloses a piRNA biomarker related to a bladder cancer and application thereof. The biomarker comprises 197 piRNAs, wherein the promoted 106 piRNAs are shown in SEQ ID No.1-106; the lowered 91 piRNAs are shown in SEQ ID No.107-197. By adopting the piRNA biomarker, the condition that the piRNA biomarker can be used as an important biological detection index in early clinical differential diagnosis of the bladder cancer is found out for the first time; a theoretical foundation is provided for research of the bladder cancer piRNA in future; a new idea is provided for molecular diagnosis of the bladder cancer; the piRNA biomarker has important theoretical significance and potential use value.

Description

PiRNA biomarker and the application thereof relevant to bladder cancer
Technical field
The invention belongs to biotechnology and oncology, especially relate to the detection method of piRNA biomarker in Bladder Cancer and expression amount thereof.
Background technology:
Bladder cancer is one of modal malignant tumour of urinary system, the whole world, and bladder cancer ranks the 7th in male tumor, ranks the 17 in women.In China's urologic neoplasms, no matter bladder cancer is that sickness rate or mortality ratio all rank first and sickness rate is the trend that raises year by year, and most of patient age is between 50-60 between year.Bladder cancer more than 90% is transitional epithelial cell cancer, by pathological staging, can be divided into superficial bladder cancer (in early days) and flesh layer invasive bladder cancer (progressive stage).Developing of bladder cancer, can have a strong impact on patient's existence and quality of life, therefore find the responsive special biological markers that can be used for early diagnosis, to reaching, utilize it to carry out prevention, screening and diagnosis, thereby improve existence and the quality of life of bladder cancer patients.
The research of RNA enjoys the extensive concern of scientific circles in recent years, especially some noncoding little RNA(non-coding RNA, ncRNA) molecule, these ncRNA molecules can regulate the important physiological process of Growth of Cells and growth.The vital process of ncRNA regulating cell comprises the gene silencing of transcriptional level and post-transcriptional level.General little ncRNA can be divided three classes: siRNAs, microRNAs(miRNAs) and PIWI-interacting RNAs(PIWI albumen correlative coding RNAs, be called for short piRNAs) [2].Wherein the research of piRNA is fewer, and the function that piRNA is potential and biogenesis exist obviously different from miRNA and siRNA.The about 26nt – of piRNA length 31nt, can form piRNA mixture (piRC) and bring into play its biological function with PIWI protein binding.Be different from miRNA, piRNA is sheared and is produced by the precursor containing loop-stem structure.The genome of piRNA sequence distributes and shows, the common cluster of piRNA occurs, and has very strong positive-sense strand or antisense strand specificity.
For the generation mechanism of piRNA, there is no at present direct biological chemistry evidence.But one has proposed " table tennis model " hypothesis based on bioinformatic analysis.This model is thought: the piRNA precursor of antisense strand is through the processing of some unknown nuclease, generated 5 ' end and there is U(uridylic) the elementary piRNA of preferences, these piRNA and Aub or Piwi protein binding form piRC, piRC is under the guiding of piRNA, the mode of matching by base complementrity is identified and in conjunction with the piRNA precursor of positive-sense strand, then positive-sense strand piRNA precursor is sheared to cutting, produce the 5 ' end of secondary piRNA, then this secondary piRNA is formed 3 ' end by certain nuclease cutting, finally generates ripe positive-sense strand piRNA.Positive-sense strand piRNA and Ago3(PIWI) in conjunction with forming piRC, by similar mode, identify and cut the piRNA precursor of antisense strand.So just formed a piRNA biology and circulated, the mutual identification of positive-sense strand and antisense strand, cuts by the shearing of piRC, and circulation produces piRNA.Be somebody's turn to do at present " table tennis model " hypothesis, obtained many scholars' extensive approval.
It should be noted that, Nature magazines in July, 2006, two independently seminar delivered the article about piRNA the same period, the method for the utilization Northern blot such as Aravin confirms to only have in sexual cell and just has piRNA, while Girard etc. has also proved this existence form.Recently, also have many research to report the dependency between piRNA and human tumor, but the research of machine-processed aspect is less.As the method for the employing Solexa order-checkings such as Lu, find that piRNA exists in HeLa Cells, Chen etc. also find that piRNA is present in people's kinds of tumor cells.Chen etc. finds in the research of piRNA in addition, the clinical stages significant correlation of piRNA-651 and cancer of the stomach, observe its expression level in cancer of the stomach, colorectal carcinoma, lung cancer and breast cancer tissue simultaneously and be significantly higher than its corresponding cancer beside organism, point out this piRNA to can be used as the potential biomarker of diagnosing tumor.
Extraction and detection for piRNA in bladder body at present still lacks, and all do not relate in the other healthy tissues of Bladder Cancer and corresponding cancer thereof relevant to which piRNA in another prior art.
Summary of the invention
The technical problem solving: the object of the invention is to overcome deficiency of the prior art, a kind of piRNA biomarker and application thereof for diagnosing bladder cancer is provided, set up bladder cancer patients and organize the express spectra of piRNA, disclose the value of piRNA to Diagnosis of Bladder in tissue, for bladder cancer early discovery clinically provides basis.
Technical scheme: a kind of piRNA biomarker relevant to bladder cancer, totally 197 of described biomarker: piRNA, wherein:
Raise 106, as shown in SEQ ID No.1～106: piR-52681, piR-48765, piR-36984, piR-52373, piR-31112, piR-36360, piR-61291, piR-54826, piR-51747, piR-43148, piR-45726, piR-48368, piR-58119, piR-57089, piR-39017, piR-43887, piR-37525, piR-39614, piR-39018, piR-35831, piR-39592, piR-33050, piR-34998, piR-38355, piR-56914, piR-34920, piR-37523, piR-45582, piR-51256, piR-37524, piR-52680, piR-31111, piR-56252, piR-58255, piR-31101, piR-30597, piR-32079, piR-54098, piR-34984, piR-31108, piR-34000, piR-31961, piR-33650, piR-57293, piR-33470, piR-36475, piR-47132, piR-45029, piR-58050, piR-36707, piR-60668, piR-34756, piR-47161, piR-55637, piR-44190, piR-31013, piR-50437, piR-33543, piR-43217, piR-38376, piR-50657, piR-61404, piR-51364, piR-48118, piR-30327, piR-54136, piR-45459, piR-44085, piR-52697, piR-49813, piR-57447, piR-39232, piR-57132, piR-39763, piR-45051, piR-33043, piR-53177, piR-43105, piR-57140, piR-40110, piR-37972, piR-40982, piR-42947, piR-43876, piR-38354, piR-57141, piR-57567, piR-44956, piR-33368, piR-56037, piR-57143, piR-43107, piR-33733, piR-57142, piR-57139, piR-34534, piR-56276, piR-32683, piR-55475, piR-34532, piR-33730, piR-59160, piR-61324, piR-49600, piR-52323, piR-38240,
Lower 91, as shown in SEQ ID No.107～197: piR-60152, piR-34373, piR-30504, piR-34374, piR-55023, piR-34375, piR-43452, piR-52155, piR-34380, piR-34379, piR-44265, piR-40154, piR-34420, piR-48578, piR-35688, piR-56066, piR-37243, piR-47995, piR-49208, piR-34377, piR-56061, piR-34376, piR-38145, piR-58986, piR-30506, piR-37988, piR-33851, piR-39786, piR-56470, piR-43801, piR-45852, piR-58572, piR-49842, piR-49039, piR-51517, piR-35536, piR-35924, piR-33814, piR-47258, piR-32538, piR-30436, piR-52398, piR-36439, piR-34177, piR-52399, piR-35359, piR-31167, piR-56951, piR-42797, piR-40163, piR-31440, piR-32499, piR-34422, piR-31133, piR-36614, piR-45571, piR-32406, piR-46447, piR-34348, piR-31252, piR-45112, piR-54110, piR-58451, piR-49939, piR-49552, piR-39004, piR-30769, piR-40571, piR-50830, piR-45655, piR-42221, piR-35166, piR-35167, piR-39572, piR-39516, piR-38856, piR-35717, piR-33417, piR-38060, piR-55800, piR-33401, piR-39593, piR-35762, piR-53013, piR-35899, piR-34726, piR-33466, piR-34194, piR-54213, piR-58463, piR-34366.
A piRNA biomarker relevant to bladder cancer, described biomarker is the piR-52681 of rise and the piR-60152 of downward.
The application of described piRNA biomarker in preparation bladder cancer detection kit.
The detection of the piRNA biomarker relevant to bladder cancer and express spectra establishment method, comprise the following steps:
(1) use Trizol reagent (Invitrogen) to extract total RNA from the cancer beside organism of the cancerous tissue of bladder cancer patients and correspondence thereof, by the piRNA cDNA microarray of Array Star, go out the piRNA of candidate's differential expression;
(2) RNA reverses, and adopts miScript reverse transcription test kit (Qiagen) to carry out reverse transcription reaction to RNA extracting solution, obtains cDNA solution;
(3) PCR detects, and uses miScript SYBR Green PCR detection kit to carry out pcr amplification reaction to the cDNA solution in step (2), and then completes the checking to candidate piRNA, sets up bladder cancer piRNA express spectra.
The detection method of the piRNA biomarker expression amount relevant to bladder cancer, the method for employing SYBR qRT-PCR detects the expression amount of piRNA in bladder body:
(1) reverse transcription: adopt miScript reverse transcription test kit (Qiagen) to carry out reverse transcription reaction to the total RNA extracting, concrete operations are carried out according to the specification sheets of this test kit, first in 0.5mL centrifuge tube, add 5 μ L DEPC distilled waters, then add 1 μ L reverse transcription mix reagent and 4 μ L RT damping fluids, the total RNA that finally adds 10 μ L0.1 μ g/ μ L, total reaction system 20 μ L, the reaction conditions of the method is: 37 ℃ are reacted 1 hour, 95 ℃ are reacted 5 minutes, obtain cDNA solution;
(2) piR-52681 real-time fluorescence quantitative PCR detects: adopt miScript SYBR Green PCR detection kit to carry out pcr amplification reaction to the cDNA solution obtaining in step (1), amplimer is the general downstream primer of little RNA that piR-52681 amplification upstream primer and PCR detect, with ABI7900HT Real-Time PCR instrument, detect again, concrete operations are carried out according to this test kit and quantitative real time PCR Instrument specification sheets, reaction system comprises: the general downstream primer of little RNA that 1 μ L piR-52681 amplification upstream primer and 1 μ LPCR detect, 5 μ L SYBR Green PCR reaction buffers, 2 μ L DEPC distilled waters and 1 μ L0.1 μ g/ μ L cDNA diluent, the reaction conditions of the method: 95 ℃ of denaturations 15 minutes, 94 ℃ of sex change 15 seconds, anneal 30 seconds for 60 ℃, 70 ℃ are extended 30 seconds, the stage of 94 ℃-70 ℃ totally 40 circulations,
(3) piR-60152 real-time fluorescence quantitative PCR detects: identical with the method in step (2), the difference just upstream primer of amplification replaces piR-60152 by piR-52681 specific amplification upstream primer, detects the expression level that obtains piR-60152 in Bladder Cancer and cancer beside organism;
(4) U6 real-time fluorescence quantitative PCR detects: identical with the method in step (2), the difference just upstream primer of amplification has piR-52681 specific amplification upstream primer to replace U6, detects the expression level that obtains U6 in Bladder Cancer and cancer beside organism; In this programme, U6 is as the internal reference of piR-52681 and piR-60152.
Beneficial effect: the present invention finds that piRNA is in the clinical Early Identification diagnosis of bladder cancer first, can be used as a kind of important Biological Detection index, for the research of bladder cancer piRNA from now on provides theoretical foundation, and for molecular level diagnosing bladder cancer provides new thinking, there is great theory significance and potential use value.
Accompanying drawing explanation
Fig. 1 is that real-time fluorescence quantitative PCR detects the expression of piR-52681 in Bladder Cancer and cancer beside organism;
Fig. 2 is that real-time fluorescence quantitative PCR detects the expression of piR-60152 in Bladder Cancer and cancer beside organism.
Embodiment
Below in conjunction with embodiment, the present invention will be described in detail: the present embodiment is implemented under the prerequisite with technical solution of the present invention, provided detailed embodiment and process, but the scope that comprises of the present invention is not limited to following embodiment.In the following examples, not marked condition and method etc. are all carried out according to routine.
Embodiment 1: the extraction of RNA in the collection of sample and tissue
Collect the other healthy tissues of 19 pairs of new bladder cancer and corresponding cancer thereof.Tissue-derived year June in January, 2010 to 2012, and through Hospital Pathological Department, diagnosis is confirmed, all research objects are all signed Informed Consent Form.
Use Trizol method to extract the total RNA in tissue: the Bladder Cancer or the other healthy tissues of cancer that take 100mg are put in glass grinding device, then add liquid nitrogen grinding to tissue to become dry powder, then dry powder is transferred in the centrifuge tube that 1.5mL removes RNA enzyme (having added in advance 1mL Trizol solution in pipe) with sleaker, put upside down and mix 10 times, standing 5 minutes of room temperature; In every 1mL Trizol, add 0.2mL chloroform, cover tightly sample hose lid, with exert oneself shaking test tube 15 seconds of hand, it is fully mixed, room temperature centrifugal 15 minutes of 12000g after standing 5 minutes; Upper strata water is proceeded in the centrifuge tube that new 1.5mL removes RNA enzyme (the about 0.4-0.5mL of supernatant amount), add 0.5mL Virahol, be put in-20 ℃ 1 hour after mixing, then 12000g is centrifugal 10 minutes; Carefully outwell supernatant liquor, leave and take the precipitation of centrifuge tube bottom, add ethanol (precooling) the vibration washing RNA precipitation of the 75%wt that 1mL now joins once, centrifugal 5 minutes of last 7500g; Carefully outwell supernatant, then the centrifuge tube with precipitation is placed in to the Bechtop machine that blows in and dries up (approximately 30 minutes, now RNA precipitation bleach); The DEPC water dissolution that adds 20 μ L in centrifuge tube can be hatched 10 minutes hydrotropies at 55-60 ℃.The total RNA having extracted is stored in the Ultralow Temperature Freezer of-70 ℃, or immediately for reverse transcription.Total RNA mass analysis: by the detection of RNA A230, A260 and A280 (A260/A280=1.8-2.1, A260/A230>1.80; ), and total RNA is adopted to agarose gel electrophoresis, according to the quality of the total RNA of ratio Analysis of 28S rRNA and 18S rRNA.
The investigation of sample epidemiology and clinical data
All patients' bladder body is all through the pathological diagnosis of two pathologist, do not suffer from other tumour before including this research in, and without radiation or chemotherapy.Investigation content comprises general demographic characteristics, clinical data, smoking history, history of drinking history, diet, personal history and family history etc.Smoker is defined as smoking every day and surpasses 1 and lasting more than 1 year, and accumulative total smoking capacity " bag year " calculates by (smoking capacity/20 every day) * years of smoking.Alcohol user is defined as at least 3 times weekly, continues more than 1 year.
PiRNA express spectra is for screening the candidate piRNA of the other healthy tissues differential expression of bladder cancer and corresponding cancer thereof
Randomly draw the other healthy tissues of 3 pairs of cancers and cancer, 23677 piRNAs that adopted people piRNA chip detection.Results of statistical analysis shows: it is 197 that the above and P value of 2 times of differential expressions is less than 0.05 piRNA gene number, wherein 106 of rise, and 91 (table 1) of downward.We have listed differential expression rise, have lowered the most significant front ten piRNAs(tables 2).
The detection method of piRNA expression amount in the other healthy tissues of Bladder Cancer and cancer thereof
The method of employing SYBR qRT-PCR detects the expression amount of piRNA in bladder body
(1) reverse transcription: adopt miScript reverse transcription test kit (Qiagen) to carry out reverse transcription reaction to the total RNA extracting, concrete operations are carried out according to the specification sheets of this test kit.First in 0.5mL centrifuge tube, add 5 μ L DEPC distilled waters, then add 1 μ L reverse transcription mix reagent and 4 μ L RT damping fluids, finally add total RNA of 10 μ L0.1 μ g/ μ L, total reaction system 20 μ L.The reaction conditions of the method is: 37 ℃ are reacted 1 hour, and 95 ℃ are reacted 5 minutes, obtain cDNA solution.
(2) piR-52681 real-time fluorescence quantitative PCR detects: adopt miScript SYBR Green PCR detection kit to carry out pcr amplification reaction to the cDNA solution obtaining in (1), amplimer is the general downstream primer of little RNA that piR-52681 amplification upstream primer and PCR detect, with ABI7900HT Real-Time PCR instrument, detect, concrete operations are carried out according to this test kit and quantitative real time PCR Instrument specification sheets again.Reaction system comprises: the general downstream primer of little RNA that 1 μ L piR-52681 amplification upstream primer and 1 μ LPCR detect, 5 μ L SYBR Green PCR reaction buffers, 2 μ L DEPC distilled waters and 1 μ L cDNA diluent (0.1 μ g/ μ L).The reaction conditions of the method: 95 ℃ of denaturations 15 minutes, 94 ℃ of sex change 15 seconds, 60 ℃ of annealing 30 seconds, 70 ℃ are extended 30 seconds, the stage of 94 ℃-70 ℃ totally 40 circulations.
(3) piR-60152 real-time fluorescence quantitative PCR detects: basic identical with the method in (2), the difference just upstream primer of amplification replaces piR-60152 by piR-52681 specific amplification upstream primer, detects the expression level that obtains piR-60152 in Bladder Cancer and cancer beside organism.
(4) U6 real-time fluorescence quantitative PCR detects: basic identical with the method in (2), the difference just upstream primer of amplification has piR-52681 specific amplification upstream primer to replace U6, detects the expression level that obtains U6 in Bladder Cancer and cancer beside organism.U6 is as the internal reference of piR-52681 and piR-60152 in this experiment.
Analyze piR-52681(DQ585569) and the piR-60152(DQ594040) diagnostic value to bladder cancer
In example, the expression of piRNA is to represent with CT value.CT value represents that a kind of object piRNA molecule reaches the number of times of the needed PCR circulation of experimental design threshold value.In the situation that object piR-52681, piR-60152 are identical with internal reference U6 amplification efficiency, can directly obtain the quantitative Δ CT=CT that object piR-52681, piR-60152 are equivalent to internal reference object-CT internal reference.Adopt 2 -Δ CTrepresent that object piRNA expresses the multiple of expressing variation with respect to internal reference U6.All statistical tests are bilateral probability inspection, and P<0.05 is that difference has statistical significance.Adopt SPSS13.0 software to do further statistical study.
Statistic analysis result discovery, the expression of piR-52681 in Bladder Cancer is significantly higher than the other healthy tissues of its corresponding cancer, and (cancerous tissue is compared with the other healthy tissues of cancer: 7.58 ± 7.49versus2.91 ± 1.33, P=0.026) (Fig. 1).Significantly lower than the other healthy tissues of its corresponding cancer, (cancerous tissue is compared with the other healthy tissues of cancer: 3.95 ± 2.05versus8.82 ± 7.81, P=0.027) (Fig. 2) in the expression of piR-60152 in Bladder Cancer.
Difference piRNA express spectra in the other healthy tissues of table 1. bladder cancer and cancer
Figure BDA0000402925290000061
Continuous upper table
Figure BDA0000402925290000071
Continuous upper table
Figure BDA0000402925290000081
Continuous upper table
Figure BDA0000402925290000091
Continuous upper table
Figure BDA0000402925290000101
ancbi database
Table 2. differential expression raises, lowers the most significant front ten piRNAs
Figure BDA0000402925290000111
ancbi database
Above-mentioned example is only explanation technical conceive of the present invention and feature, and its object is to allow person skilled in the art can understand content of the present invention and implement according to this, can not limit the scope of the invention with this.All equivalent transformations that spirit is done according to the present invention or modification, within all should being encompassed in protection scope of the present invention.
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<212> DNA
<213> artificial sequence
<400> 45
cggctgttaa ccgaaaggtt ggtggt 26
<210> 46
<211> 26
<212> DNA
<213> artificial sequence
<400> 46
gtcagggaca tttctgaagc gagcgt 26
<210> 47
<211> 26
<212> DNA
<213> artificial sequence
<400> 47
tgaagcagag gcaatgaatt acgaag 26
<210> 48
<211> 28
<212> DNA
<213> artificial sequence
<400> 48
tcgctggttc gaatccggct cggaggac 28
<210> 49
<211> 29
<212> DNA
<213> artificial sequence
<400> 49
tgtcacgtgt gtggcaagat gctgagctc 29
<210> 50
<211> 31
<212> DNA
<213> artificial sequence
<400> 50
gttaagatgg cagagcccgg taatcgcata a 31
<210> 51
<211> 30
<212> DNA
<213> artificial sequence
<400> 51
ttcgatgaag agatgatgac gagtctgact 30
<210> 52
<211> 30
<212> DNA
<213> artificial sequence
<400> 52
gaggagccac tgtggcgaca gaatgctagc 30
<210> 53
<211> 27
<212> DNA
<213> artificial sequence
<400> 53
tgaagctgca gaaccaacga ggtggcc 27
<210> 54
<211> 27
<212> DNA
<213> artificial sequence
<400> 54
tgggaagtac gtcctggggt acaagca 27
<210> 55
<211> 32
<212> DNA
<213> artificial sequence
<400> 55
tcctaggact gaatcaccac cccagaagag ca 32
<210> 56
<211> 30
<212> DNA
<213> artificial sequence
<400> 56
agcacatgat gattccagcg tgcgtctgaa 30
<210> 57
<211> 27
<212> DNA
<213> artificial sequence
<400> 57
tgcaatggca tgatctcggc tcactgc 27
<210> 58
<211> 32
<212> DNA
<213> artificial sequence
<400> 58
cgtgctgggc ccataaccca gaggtcgatg ga 32
<210> 59
<211> 30
<212> DNA
<213> artificial sequence
<400> 59
tcccacgtgg ctgagactac aggtgcttgc 30
<210> 60
<211> 30
<212> DNA
<213> artificial sequence
<400> 60
tacctgaaga tgaaagggga ctactaccgc 30
<210> 61
<211> 28
<212> DNA
<213> artificial sequence
<400> 61
tgcagaaagc tctgggtgtg cggcagta 28
<210> 62
<211> 28
<212> DNA
<213> artificial sequence
<400> 62
ttggaggatg aaacaaagga atctgact 28
<210> 63
<211> 30
<212> DNA
<213> artificial sequence
<400> 63
tgccaccaat gtcgagcagg cgttcatgac 30
<210> 64
<211> 27
<212> DNA
<213> artificial sequence
<400> 64
tgactgtgct gtcctgattg ttgctgc 27
<210> 65
<211> 29
<212> DNA
<213> artificial sequence
<400> 65
acaaaaaatt acccgggcgt ggtggcagg 29
<210> 66
<211> 26
<212> DNA
<213> artificial sequence
<400> 66
tggagatcct ggagctgtac tgtgac 26
<210> 67
<211> 30
<212> DNA
<213> artificial sequence
<400> 67
tctcaaagtg ctgggattac aggcgtgagc 30
<210> 68
<211> 30
<212> DNA
<213> artificial sequence
<400> 68
tcctaaagtg ctggggttac aggcatgagc 30
<210> 69
<211> 31
<212> DNA
<213> artificial sequence
<400> 69
tgctagggtc acctgtaccc caggctggag c 31
<210> 70
<211> 28
<212> DNA
<213> artificial sequence
<400> 70
tgatgagacg aagttgacgc tgcatgga 28
<210> 71
<211> 26
<212> DNA
<213> artificial sequence
<400> 71
tggtggatgc gcacactgcc gaagtc 26
<210> 72
<211> 30
<212> DNA
<213> artificial sequence
<400> 72
tagcagctgt atcgggcttc ctcatcctgc 30
<210> 73
<211> 30
<212> DNA
<213> artificial sequence
<400> 73
tggtctcaaa ctcctgacct caggtgatct 30
<210> 74
<211> 31
<212> DNA
<213> artificial sequence
<400> 74
tagggtcacc tgtaccccag gctggagctg c 31
<210> 75
<211> 27
<212> DNA
<213> artificial sequence
<400> 75
tcggaacctg cagacacttg tggagga 27
<210> 76
<211> 27
<212> DNA
<213> artificial sequence
<400> 76
ccccccactg ctaaatttga ctggcta 27
<210> 77
<211> 30
<212> DNA
<213> artificial sequence
<400> 77
tgctgggatg acaggcatga gccactgcgc 30
<210> 78
<211> 28
<212> DNA
<213> artificial sequence
<400> 78
tcccaaagtg ctggaattac aggcgtga 28
<210> 79
<211> 29
<212> DNA
<213> artificial sequence
<400> 79
tggtctcgaa ctcctgacct caggtgatc 29
<210> 80
<211> 28
<212> DNA
<213> artificial sequence
<400> 80
tagtttcaga aggaatggta ccagctcc 28
<210> 81
<211> 26
<212> DNA
<213> artificial sequence
<400> 81
tacagcacac tgatgggtct tgactc 26
<210> 82
<211> 29
<212> DNA
<213> artificial sequence
<400> 82
tcacaaagat gagtggtgaa aatctgatc 29
<210> 83
<211> 32
<212> DNA
<213> artificial sequence
<400> 83
tccatcgtta caatggcctc tttagaccca gc 32
<210> 84
<211> 31
<212> DNA
<213> artificial sequence
<400> 84
tccgagtgca gaccacgccg gactacagcc c 31
<210> 85
<211> 30
<212> DNA
<213> artificial sequence
<400> 85
tacctcatga agatcctcac cgagcgcggc 30
<210> 86
<211> 30
<212> DNA
<213> artificial sequence
<400> 86
tggtctcgaa ctcctgacct caggtgatcc 30
<210> 87
<211> 30
<212> DNA
<213> artificial sequence
<400> 87
tggtgtgatc tcggctcact gcaacctcct 30
<210> 88
<211> 30
<212> DNA
<213> artificial sequence
<400> 88
tcgcaatcca actggtacac tggcatgagt 30
<210> 89
<211> 30
<212> DNA
<213> artificial sequence
<400> 89
cgatcaagca gccgtgcaga gatgtgcctc 30
<210> 90
<211> 29
<212> DNA
<213> artificial sequence
<400> 90
tgggattaca ggcatgagcc accgcgcct 29
<210> 91
<211> 30
<212> DNA
<213> artificial sequence
<400> 91
tggtctcgaa ctcctgacct caggtgatct 30
<210> 92
<211> 30
<212> DNA
<213> artificial sequence
<400> 92
tcccaaagtg ctgggaatac aggcatgagc 30
<210> 93
<211> 30
<212> DNA
<213> artificial sequence
<400> 93
ctcgaactcc tgacctcagg tgatctgcct 30
<210> 94
<211> 31
<212> DNA
<213> artificial sequence
<400> 94
tggtctcgaa ctcctgacct caggtgatcc a 31
<210> 95
<211> 26
<212> DNA
<213> artificial sequence
<400> 95
tggtctcgaa ctcctgacct caggta 26
<210> 96
<211> 29
<212> DNA
<213> artificial sequence
<400> 96
gagaaagctc acaagaactg ctaactcat 29
<210> 97
<211> 28
<212> DNA
<213> artificial sequence
<400> 97
tgggctgcgg tggccagggc cgtgagtc 28
<210> 98
<211> 26
<212> DNA
<213> artificial sequence
<400> 98
catttgcggc cggcgccagg gtggag 26
<210> 99
<211> 28
<212> DNA
<213> artificial sequence
<400> 99
tgggaacctg cagacacttg tggaggag 28
<210> 100
<211> 30
<212> DNA
<213> artificial sequence
<400> 100
gagaaagctc acaagaactg ctaactcaca 30
<210> 101
<211> 28
<212> DNA
<213> artificial sequence
<400> 101
ctcctgaagt cagcgagacc acgaaccc 28
<210> 102
<211> 26
<212> DNA
<213> artificial sequence
<400> 102
tgtgtgctaa atgtgttcgt gacagg 26
<210> 103
<211> 26
<212> DNA
<213> artificial sequence
<400> 103
ttggaacaac cactgagcta cggaga 26
<210> 104
<211> 30
<212> DNA
<213> artificial sequence
<400> 104
tgatcaagca gccgtgcaga gatgtgcctc 30
<210> 105
<211> 30
<212> DNA
<213> artificial sequence
<400> 105
tgcctttgct gatctgacag gaggcagagc 30
<210> 106
<211> 30
<212> DNA
<213> artificial sequence
<400> 106
taccatcttg gctcactgca acctccgcct 30
<210> 107
<211> 31
<212> DNA
<213> artificial sequence
<400> 107
ttcaagccag gaaagcactg tagctccccc a 31
<210> 108
<211> 27
<212> DNA
<213> artificial sequence
<400> 108
gaccaatgat gagacagtgt ttatgaa 27
<210> 109
<211> 26
<212> DNA
<213> artificial sequence
<400> 109
accaatgatg agacagtgtt tatgaa 26
<210> 110
<211> 28
<212> DNA
<213> artificial sequence
<400> 110
gaccaatgat gagacagtgt ttatgaaa 28
<210> 111
<211> 30
<212> DNA
<213> artificial sequence
<400> 111
tggcccctta cccttccaga gatgaagccc 30
<210> 112
<211> 28
<212> DNA
<213> artificial sequence
<400> 112
gaccaatgat gagacagtgt ttatgaac 28
<210> 113
<211> 31
<212> DNA
<213> artificial sequence
<400> 113
tccccaaaac aagcacagta gcctgcacag t 31
<210> 114
<211> 26
<212> DNA
<213> artificial sequence
<400> 114
tgcctgatga tctgagatgg aacggt 26
<210> 115
<211> 31
<212> DNA
<213> artificial sequence
<400> 115
gaccaatgat gagtattctg gggtgtctga a 31
<210> 116
<211> 30
<212> DNA
<213> artificial sequence
<400> 116
gaccaatgat gagtattctg gggtgtctga 30
<210> 117
<211> 26
<212> DNA
<213> artificial sequence
<400> 117
tcctcacacc gcacggcagg cacaga 26
<210> 118
<211> 29
<212> DNA
<213> artificial sequence
<400> 118
tataccgaga cattccattg cccagggac 29
<210> 119
<211> 31
<212> DNA
<213> artificial sequence
<400> 119
gacctatgat gatgactggt ggcgtatgag t 31
<210> 120
<211> 31
<212> DNA
<213> artificial sequence
<400> 120
tgagatgctg atggggatct atgaacggat c 31
<210> 121
<211> 30
<212> DNA
<213> artificial sequence
<400> 121
gctgttatgc atctaccggc gcccaccctc 30
<210> 122
<211> 27
<212> DNA
<213> artificial sequence
<400> 122
tgggcaaggc agccagacag gcagtga 27
<210> 123
<211> 28
<212> DNA
<213> artificial sequence
<400> 123
taactgaact taaaagaaaa gccaggga 28
<210> 124
<211> 27
<212> DNA
<213> artificial sequence
<400> 124
tgactaatta gactgggaaa caagggc 27
<210> 125
<211> 29
<212> DNA
<213> artificial sequence
<400> 125
tgaggtgtaa gcactgtatc aagacaggt 29
<210> 126
<211> 31
<212> DNA
<213> artificial sequence
<400> 126
gaccaatgat gagattggag ggtgtctgaa t 31
<210> 127
<211> 27
<212> DNA
<213> artificial sequence
<400> 127
tgggcaagaa gcactgtctg caaggcc 27
<210> 128
<211> 30
<212> DNA
<213> artificial sequence
<400> 128
gaccaatgat gagattggag ggtgtctgaa 30
<210> 129
<211> 31
<212> DNA
<213> artificial sequence
<400> 129
taccaatgat gagattggag ggtgtctgaa t 31
<210> 130
<211> 30
<212> DNA
<213> artificial sequence
<400> 130
tgtggcctgc aaaggaaggt aagtaggtgg 30
<210> 131
<211> 30
<212> DNA
<213> artificial sequence
<400> 131
accaatgatg agattggagg gtgtctgaat 30
<210> 132
<211> 26
<212> DNA
<213> artificial sequence
<400> 132
tacagctccc gtgtctggga agaagc 26
<210> 133
<211> 29
<212> DNA
<213> artificial sequence
<400> 133
ctgcactgta gcctgggtga ccagagtga 29
<210> 134
<211> 29
<212> DNA
<213> artificial sequence
<400> 134
taggtacaac ggattccggc gggcagagc 29
<210> 135
<211> 29
<212> DNA
<213> artificial sequence
<400> 135
tggggtcctg cacagatggg acgtggctt 29
<210> 136
<211> 31
<212> DNA
<213> artificial sequence
<400> 136
tcccttcccg acttgatgaa ggcagagtag c 31
<210> 137
<211> 32
<212> DNA
<213> artificial sequence
<400> 137
tctgagaagc cacaccaacc tcgttccggg ga 32
<210> 138
<211> 31
<212> DNA
<213> artificial sequence
<400> 138
tgtgagatgg agcaagatga aggccacatg t 31
<210> 139
<211> 27
<212> DNA
<213> artificial sequence
<400> 139
tgatgatcct gagatgagac caaactg 27
<210> 140
<211> 26
<212> DNA
<213> artificial sequence
<400> 140
tgaggccccg ggttcgatcc ccggca 26
<210> 141
<211> 30
<212> DNA
<213> artificial sequence
<400> 141
tgccagggaa acagtctcaa ttttacactt 30
<210> 142
<211> 29
<212> DNA
<213> artificial sequence
<400> 142
gcggtgtgac gagactcgcg tgggaagaa 29
<210> 143
<211> 28
<212> DNA
<213> artificial sequence
<400> 143
ggcacagagc aggctaggtc tgccgcga 28
<210> 144
<211> 32
<212> DNA
<213> artificial sequence
<400> 144
ctgagaagcc acaccaacct cgttccgggg at 32
<210> 145
<211> 29
<212> DNA
<213> artificial sequence
<400> 145
tgaagttggg gccgagcaag aaagtgcga 29
<210> 146
<211> 30
<212> DNA
<213> artificial sequence
<400> 146
caggctaatg ggaaacggag caaccacatg 30
<210> 147
<211> 26
<212> DNA
<213> artificial sequence
<400> 147
acagcagcca gggatagcga aggtgg 26
<210> 148
<211> 26
<212> DNA
<213> artificial sequence
<400> 148
tgcgcaggaa gactgagatg aggacc 26
<210> 149
<211> 29
<212> DNA
<213> artificial sequence
<400> 149
gtaggatgaa aacactgggg ctgaaatgc 29
<210> 150
<211> 30
<212> DNA
<213> artificial sequence
<400> 150
gaagatggag cagttgctag agacgatggt 30
<210> 151
<211> 29
<212> DNA
<213> artificial sequence
<400> 151
tgcgcaggaa gactgagatg aggacccta 29
<210> 152
<211> 29
<212> DNA
<213> artificial sequence
<400> 152
gccatctgag gtgtcgacag agaaagaga 29
<210> 153
<211> 28
<212> DNA
<213> artificial sequence
<400> 153
agctgaaaaa ggagaaaggg tcactgag 28
<210> 154
<211> 30
<212> DNA
<213> artificial sequence
<400> 154
tggtaggaat ggatgatgat gctgcagagg 30
<210> 155
<211> 31
<212> DNA
<213> artificial sequence
<400> 155
tccagggaga aaaataaaag gaagccatag a 31
<210> 156
<211> 28
<212> DNA
<213> artificial sequence
<400> 156
tatagagaaa gaaataaggg gacccggg 28
<210> 157
<211> 31
<212> DNA
<213> artificial sequence
<400> 157
aggggaaaca caggaagtgt ttaaggcatg c 31
<210> 158
<211> 31
<212> DNA
<213> artificial sequence
<400> 158
caggagagta ggatgaaaac actggggctt t 31
<210> 159
<211> 30
<212> DNA
<213> artificial sequence
<400> 159
gacctcaagg gaatgtagag aagcctcagg 30
<210> 160
<211> 27
<212> DNA
<213> artificial sequence
<400> 160
agcgcaggaa gactgagatg aggaccc 27
<210> 161
<211> 31
<212> DNA
<213> artificial sequence
<400> 161
gtggaaagct gagtaactgc tgagtaggtg t 31
<210> 162
<211> 30
<212> DNA
<213> artificial sequence
<400> 162
tctcatggga gtgcttacca atgtgtggcc 30
<210> 163
<211> 30
<212> DNA
<213> artificial sequence
<400> 163
cagcagagca ggcacagcgg tgtgacgaga 30
<210> 164
<211> 31
<212> DNA
<213> artificial sequence
<400> 164
tcttgaggga cgctagaggt agtagatgtg t 31
<210> 165
<211> 30
<212> DNA
<213> artificial sequence
<400> 165
gacatgcata gagggaagac caggtgaaga 30
<210> 166
<211> 27
<212> DNA
<213> artificial sequence
<400> 166
aggagaagta gaaggggctg cacatgc 27
<210> 167
<211> 29
<212> DNA
<213> artificial sequence
<400> 167
tcggcagagc aggcacagag gtgtgacga 29
<210> 168
<211> 26
<212> DNA
<213> artificial sequence
<400> 168
tggagacaca ggacgttctg agatgg 26
<210> 169
<211> 26
<212> DNA
<213> artificial sequence
<400> 169
tgtgaaggaa agtcaccaca gggcaa 26
<210> 170
<211> 29
<212> DNA
<213> artificial sequence
<400> 170
tgatggagag ccctgtgctg agatgaaat 29
<210> 171
<211> 26
<212> DNA
<213> artificial sequence
<400> 171
tgatagtgga actaaaatcg ggggca 26
<210> 172
<211> 30
<212> DNA
<213> artificial sequence
<400> 172
tagagagtca gaagctctat atgcgtagtg 30
<210> 173
<211> 30
<212> DNA
<213> artificial sequence
<400> 173
actgaatgaa aagaggtggc tgaggaaagc 30
<210> 174
<211> 31
<212> DNA
<213> artificial sequence
<400> 174
tattgagtgg aagtagctag ggtctcaggg g 31
<210> 175
<211> 28
<212> DNA
<213> artificial sequence
<400> 175
tgcaggacaa agacaatctg gctaggga 28
<210> 176
<211> 29
<212> DNA
<213> artificial sequence
<400> 176
tctcctgatg ccttgttatg aacagggag 29
<210> 177
<211> 28
<212> DNA
<213> artificial sequence
<400> 177
tccaagaaat actgggagtg ctggaccc 28
<210> 178
<211> 26
<212> DNA
<213> artificial sequence
<400> 178
gcagagcaag aaaagcagag atgtgg 26
<210> 179
<211> 31
<212> DNA
<213> artificial sequence
<400> 179
gcagagcagg cacagcggtg cgacgagatg t 31
<210> 180
<211> 29
<212> DNA
<213> artificial sequence
<400> 180
taggagtacg gtctaggcaa aggcatggt 29
<210> 181
<211> 26
<212> DNA
<213> artificial sequence
<400> 181
taggaccaga gatggagaat gacagc 26
<210> 182
<211> 30
<212> DNA
<213> artificial sequence
<400> 182
tagaaggtga gatctggagt taaacatgtc 30
<210> 183
<211> 26
<212> DNA
<213> artificial sequence
<400> 183
ggaaaaaaag caagatgcct caatgc 26
<210> 184
<211> 28
<212> DNA
<213> artificial sequence
<400> 184
cgcgtgggaa cgagaagaca ctcgtgga 28
<210> 185
<211> 28
<212> DNA
<213> artificial sequence
<400> 185
tacatcatgg accaaaggac agacaggc 28
<210> 186
<211> 28
<212> DNA
<213> artificial sequence
<400> 186
tgggagaaaa gccaagcaag tgctgaac 28
<210> 187
<211> 27
<212> DNA
<213> artificial sequence
<400> 187
cgccaaagga tagaaccgtc aggggcc 27
<210> 188
<211> 28
<212> DNA
<213> artificial sequence
<400> 188
taggatgaaa acactggggc tgaaatgc 28
<210> 189
<211> 30
<212> DNA
<213> artificial sequence
<400> 189
ggaactcggc caggtgctga tcggtgaggt 30
<210> 190
<211> 26
<212> DNA
<213> artificial sequence
<400> 190
tgctgagatg gagaagaccg ggggca 26
<210> 191
<211> 29
<212> DNA
<213> artificial sequence
<400> 191
ggcaaactgg tggagatgtg aactgaggc 29
<210> 192
<211> 29
<212> DNA
<213> artificial sequence
<400> 192
gaggaaggca gaacaaagga aataagaag 29
<210> 193
<211> 28
<212> DNA
<213> artificial sequence
<400> 193
cggcggtggc ggcggcggcg gcgggacc 28
<210> 194
<211> 30
<212> DNA
<213> artificial sequence
<400> 194
gaagctatgg gagataagat ggacttcctt 30
<210> 195
<211> 30
<212> DNA
<213> artificial sequence
<400> 195
tggaggaact caccttccag ggtgtcaatg 30
<210> 196
<211> 31
<212> DNA
<213> artificial sequence
<400> 196
tgtgaagttg cccaccccca tgcagagcct t 31
<210> 197
<211> 29
<212> DNA
<213> artificial sequence
<400> 197
gaccaaggag ttcatcttct cagagctgc 29

Claims (5)

1. a piRNA biomarker relevant to bladder cancer, is characterized in that, totally 197 of described biomarker: piRNA, wherein,
Raise 106, as shown in SEQ ID No.1 ~ 106: piR-52681, piR-48765, piR-36984, piR-52373, piR-31112, piR-36360, piR-61291, piR-54826, piR-51747, piR-43148, piR-45726, piR-48368, piR-58119, piR-57089, piR-39017, piR-43887, piR-37525, piR-39614, piR-39018, piR-35831, piR-39592, piR-33050, piR-34998, piR-38355, piR-56914, piR-34920, piR-37523, piR-45582, piR-51256, piR-37524, piR-52680, piR-31111, piR-56252, piR-58255, piR-31101, piR-30597, piR-32079, piR-54098, piR-34984, piR-31108, piR-34000, piR-31961, piR-33650, piR-57293, piR-33470, piR-36475, piR-47132, piR-45029, piR-58050, piR-36707, piR-60668, piR-34756, piR-47161, piR-55637, piR-44190, piR-31013, piR-50437, piR-33543, piR-43217, piR-38376, piR-50657, piR-61404, piR-51364, piR-48118, piR-30327, piR-54136, piR-45459, piR-44085, piR-52697, piR-49813, piR-57447, piR-39232, piR-57132, piR-39763, piR-45051, piR-33043, piR-53177, piR-43105, piR-57140, piR-40110, piR-37972, piR-40982, piR-42947, piR-43876, piR-38354, piR-57141, piR-57567, piR-44956, piR-33368, piR-56037, piR-57143, piR-43107, piR-33733, piR-57142, piR-57139, piR-34534, piR-56276, piR-32683, piR-55475, piR-34532, piR-33730, piR-59160, piR-61324, piR-49600, piR-52323, piR-38240,
Lower 91, as shown in SEQ ID No.107 ~ 197: piR-60152, piR-34373, piR-30504, piR-34374, piR-55023, piR-34375, piR-43452, piR-52155, piR-34380, piR-34379, piR-44265, piR-40154, piR-34420, piR-48578, piR-35688, piR-56066, piR-37243, piR-47995, piR-49208, piR-34377, piR-56061, piR-34376, piR-38145, piR-58986, piR-30506, piR-37988, piR-33851, piR-39786, piR-56470, piR-43801, piR-45852, piR-58572, piR-49842, piR-49039, piR-51517, piR-35536, piR-35924, piR-33814, piR-47258, piR-32538, piR-30436, piR-52398, piR-36439, piR-34177, piR-52399, piR-35359, piR-31167, piR-56951, piR-42797, piR-40163, piR-31440, piR-32499, piR-34422, piR-31133, piR-36614, piR-45571, piR-32406, piR-46447, piR-34348, piR-31252, piR-45112, piR-54110, piR-58451, piR-49939, piR-49552, piR-39004, piR-30769, piR-40571, piR-50830, piR-45655, piR-42221, piR-35166, piR-35167, piR-39572, piR-39516, piR-38856, piR-35717, piR-33417, piR-38060, piR-55800, piR-33401, piR-39593, piR-35762, piR-53013, piR-35899, piR-34726, piR-33466, piR-34194, piR-54213, piR-58463, piR-34366.
2. a piRNA biomarker relevant to bladder cancer, is characterized in that, described biomarker is the piR-52681 of rise and the piR-60152 of downward.
3. the application of the piRNA biomarker described in claim 1 or 2 in preparation bladder cancer detection kit.
4. the detection of the piRNA biomarker relevant to bladder cancer as claimed in claim 1 or 2 and an express spectra establishment method, is characterized in that comprising the following steps:
(1) use Trizol reagent (Invitrogen) to extract total RNA from the cancer beside organism of the cancerous tissue of bladder cancer patients and correspondence thereof, by the piRNA cDNA microarray of Array Star, go out the piRNA of candidate's differential expression;
(2) RNA reverses, and adopts miScript reverse transcription test kit (Qiagen) to carry out reverse transcription reaction to RNA extracting solution, obtains cDNA solution;
(3) PCR detects, and uses miScript SYBR Green PCR detection kit to carry out pcr amplification reaction to the cDNA solution in step (2), and then completes the checking to candidate piRNA, sets up bladder cancer piRNA express spectra.
5. the detection method of the piRNA biomarker expression amount relevant to bladder cancer as claimed in claim 2, is characterized in that adopting the method for SYBR qRT-PCR to detect the expression amount of piRNA in bladder body:
(1) reverse transcription: adopt miScript reverse transcription test kit (Qiagen) to carry out reverse transcription reaction to the total RNA extracting, concrete operations are carried out according to the specification sheets of this test kit, first in 0.5mL centrifuge tube, add 5 μ L DEPC distilled waters, then add 1 μ L reverse transcription mix reagent and 4 μ L RT damping fluids, the total RNA that finally adds 10 μ L 0.1 μ g/ μ L, total reaction system 20 μ L, the reaction conditions of the method is: 37 ℃ are reacted 1 hour, 95 ℃ are reacted 5 minutes, obtain cDNA solution;
(2) piR-52681 real-time fluorescence quantitative PCR detects: adopt miScript SYBR Green PCR detection kit to carry out pcr amplification reaction to the cDNA solution obtaining in step (1), amplimer is the general downstream primer of little RNA that piR-52681 amplification upstream primer and PCR detect, with ABI 7900HT Real-Time PCR instrument, detect again, concrete operations are carried out according to this test kit and quantitative real time PCR Instrument specification sheets, reaction system comprises: the general downstream primer of little RNA that 1 μ L piR-52681 amplification upstream primer and 1 μ L PCR detect, 5 μ L SYBR Green PCR reaction buffers, 2 μ L DEPC distilled waters and 1 μ L 0.1 μ g/ μ L cDNA diluent, the reaction conditions of the method: 95 ℃ of denaturations 15 minutes, 94 ℃ of sex change 15 seconds, anneal 30 seconds for 60 ℃, 70 ℃ are extended 30 seconds, the stage of 94 ℃-70 ℃ totally 40 circulations,
(3) piR-60152 real-time fluorescence quantitative PCR detects: identical with the method in step (2), the difference just upstream primer of amplification replaces piR-60152 by piR-52681 specific amplification upstream primer, detects the expression level that obtains piR-60152 in Bladder Cancer and cancer beside organism;
(4) U6 real-time fluorescence quantitative PCR detects: identical with the method in step (2), the difference just upstream primer of amplification has piR-52681 specific amplification upstream primer to replace U6, detects the expression level that obtains U6 in Bladder Cancer and cancer beside organism; In this programme, U6 is as the internal reference of piR-52681 and piR-60152.
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