CN103571954A

CN103571954A - Application of single nucleotide polymorphism sequences of TERT (telomerase reverse transcriptase) promoters to detection on neoplasms of urinary systems

Info

Publication number: CN103571954A
Application number: CN201310514819.5A
Authority: CN
Inventors: 吴松; 黄毅; 王波; 蔡志明; 梅红兵
Original assignee: Shenzhen Second Peoples Hospital
Current assignee: Shenzhen Second Peoples Hospital
Priority date: 2013-10-28
Filing date: 2013-10-28
Publication date: 2014-02-12

Abstract

The invention provides application of single nucleotide polymorphism sequences of TERT (telomerase reverse transcriptase) promoters to detection on neoplasms of urinary systems, belonging to the biotechnical field. The single nucleotide polymorphism sequences of the TERT promoters comprise a base polymorphism sequence, with C or T in the locus 1,295,228, of a TERT promoter chr5, a base polymorphism sequence, with CC or TT in the loci 1,295,242-1,295,243, of the TERT promoter chr5 and a base polymorphism sequence, with C or T in the locus 1,295,250, of the TERT promoter chr5. The neoplasms of urinary systems are bladder transitional cell carcinomas or upper urothelial cell carcinomas. The application has the advantages that high frequency mutation of the TERT promoters is found in urologic neoplasms for the first time and a new biomarker is provided for prognosis and monitoring of neoplasms of urogenital systems.

Description

The application of the single nucleotide polymorphism sequence of TERT promotor in detecting urologic neoplasms

Technical field

The invention belongs to biological technical field, the application of the single nucleotide polymorphism sequence that is specifically related to TERT promotor in detecting urologic neoplasms.

Background technology

Bladder cancer is to come the modal tumour in the whole world of the 9th.The 2013 Nian， U.S. have 73000 patients of surpassing to be diagnosed as bladder cancer, wherein death toll approximately 15000 people.Wherein surpass 90% patient and be diagnosed as at first primary bladder cancer, about 75% patient is Superficial bladder cancer at present, 20% be patient with invasive bladder tumor, 5% remaining first visit is metastatic tumo(u)r.Previous research shows, bladder cancer has changeable clinical manifestation and genetic background.8 genes involveds (UTX, MLL-MLL3, CREBBP-EP300, NCOR1, ARID1A, CHD6) have been reported in nearest tumor of bladder research.

The tumor marker that detection " bladder cancer " adopts at present mainly contains following several, but each tumor markers all exists different shortcomings:

(1), bladder tumor antigen (BTA): BTA reagent is divided into two kinds of BTA stat and BTA test; First, these two kinds of reagent all can not independently be used for making a definite diagnosis bladder cancer; In addition, BTA reagent is expensive, is still difficult at present popularize in an all-round way use;

(2), Lewis X Detection of antigen: Lewis X is a kind of abo blood group related antigen, does not have this antigen in normal urothelium; And 5%～89% transitional cell carcinoma can detect Lewis X, but the classification of Lewis X and tumour is irrelevant;

(3), NMP-22 (nuclear matrix protein22, NMP22): NMP22 is nuclear mitotic apparatus protein, the susceptibility of diagnosing bladder cancer is 48%～90%, and specificity is 70%～92%, NMP22 is to senior, and high phase bladder cancer susceptibility is higher;

(4), scleroproein/fibrin degradation product (FDP) (fibrin degradation products, FDP): the susceptibility of measuring FDP diagnosing bladder cancer in urine by tachysynthesis detection method is 68%, to the susceptibility of T2～T4 phase bladder cancer especially up to 100%;

(5), hyaluronidase detects hyaluronidase, HAase: hyaluronidase is the hyaluronic endoglycosidase of a kind of extracellular matrix degradation, in tumour progression, play an important role, application gel technique detects G2, in G3 level bladder cancer urine, hyaluronidase is active, and susceptibility reaches 92%～100%;

(6), telomerase activation (telomerase): telomere is the protective structures that is positioned at end of chromosome; with cell fission, progressively shorten; until necrocytosis; the effect of Telomerase is exactly to extend telomere; have now found that kinds of tumor cells Telomerase Activity strengthens; this method diagnosis comprises rudimentary, and lowstand tumour is in interior bladder cancer, and susceptibility can reach 91%.

Yet, cause the key point that tumor of bladder occurs not illustrated completely yet, simultaneously because the susceptibility of these individual marks is lower to such an extent as to be not applied in clinical practice the biomarker that this susceptibility that shows that needs are new is high.

Summary of the invention

In process of the test of the present invention, in surpassing 85% human tumor research, find reverse transcriptase of telomere (TERT, TERT obtains the human genomic sequence of NCBI released version 37: Feb.2009 (GRCh37/hg19) by Human Genome Browser; >hg19_ensGene_ENST00000310581_0 range=chr5:1295163-

1296562 5'pad=400 3'pad=0 strand=-repeatMasking=none) active, we think a tumorigenic important mechanisms of the mankind.TERT is the catalysed partial of Telomerase, is positioned at the short arm of a chromosome No. 5, adjustable side telomerase activity in human tumor.People apply genome sequencing and in melanoma, find TERT promoter mutation recently.People have confirmed high-frequency TERT promoter mutation in some bladder cancer samples.Some study discovery, and the activated Telomerase of tool or TERT express to increase and be associated with pathological staging and the clinical stages of tumour.Yet people also do not obtain TERT gene copy, the expression of TERT or activity remain controversial to the clinical impact of bladder cancer.In the present invention, we obtain the high frequency mutational site of TERT promotor by test, and carried out relevant external functional analysis and tested to confirm: the sudden change of these promotors can improve ETRT activity, it is the key point place that causes malignant tumour to occur, also analyzed applying clinical pathological factor and assessed TERT gene as the effect of the biomarker of early-stage cancer detection and prognosis, shown that these sudden changes have great importance to the diagnosis and prognosis of urogenital system malignant tumour.

The application of the single nucleotide polymorphism sequence that the object of the invention is to disclose TERT promotor in detecting urologic neoplasms.

The object of the invention is to be achieved through the following technical solutions:

The application of the single nucleotide polymorphism sequence of TERT promotor in detecting urologic neoplasms, the single nucleotide polymorphism sequence of described TERT promotor is:

TERT promotor chr5,1,295,228 is the nucleotide polymorphisms sequence of C or T;

TERT promotor chr5,1,295,242-1,295,243 is the nucleotide polymorphisms sequence of CC or TT;

TERT promotor chr5,1,295,250 is the nucleotide polymorphisms sequence of C or T.

The application of the single nucleotide polymorphism sequence of the TERT promotor described in technique scheme in detecting urologic neoplasms, wherein, described urologic neoplasms is transitional cell carcinoma of bladder or upper Urothelium carcinoma.

The application of the single nucleotide polymorphism sequence of the TERT promotor described in technique scheme in detecting urologic neoplasms, wherein, described application comprises the steps:

(1), from sample urinary system tissue, extract DNA;

(2), the testing gene group DNA that comprises TERT promoter gene of take is template, take primer pair P as primer, pcr amplification TERT promoter gene, described primer pair P is:

TF：5’-CAGCGCTGCCTGAAACTC-3’，

TR：5’-GTCCTGCCCCTTCACCTT-3’；

After pcr amplification completes, PCR product is carried out to sanger order-checking, while containing the single nucleotide polymorphism sequence of TERT promotor in sample, this sample is transitional cell carcinoma of bladder or upper Urothelium carcinoma sample.

The application of the single nucleotide polymorphism sequence of the TERT promotor described in technique scheme in detecting urologic neoplasms, wherein, described PCR reaction system volume is 20 μ l, contains dNTP 2 μ l, 10 * PCR Buffer, 2 μ l, TF (10 μ M) 1 μ l, TR (10 μ M) 1 μ l, template DNA 1 μ l, dH ₂o 12.5 μ l and TaKaRa Taq HS 0.5 μ l.

The application of the single nucleotide polymorphism sequence of the TERT promotor described in technique scheme in detecting urologic neoplasms, wherein, described pcr amplification reaction program is: 93 ℃ of 2 minutes denaturations, then by 93 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, totally 40 do circulation, last 72 ℃ are extended for 7 minutes.

The present invention has following beneficial effect:

1, the present invention provides a kind of new tumor markers for detecting bladder cancer, to finding that Susceptible population and the gene diagnosis of bladder cancer and relative disease thereof has important value;

2, the key point place that the high frequency of the TERT promotor in the present invention sudden change possibility bladder cancer occurs, significant to the pathogenesis of the diseases such as exploration bladder cancer;

3, the present invention, for clinical biotherapy provides molecular targeted, also opens up new treatment approach and plays an important role simultaneously.

Accompanying drawing explanation:

Fig. 1 is for carrying out somatic mutation site on anti-chain that sanger order-checking obtains by ABI 3730 XL sequenators to PCR product;

Fig. 2 is the analytical results that PCR (RT-PCR) expresses TERT, and wherein WT is that wild-type, C228T are normal chain chr5, and 1,295,228C>T sudden change and C250T are normal chain chr5,1,295,250 C>T;

Fig. 3 is the expression analysis result of protein immunization imprinting technology for detection TERT, and wherein WT is that wild-type, C228T are normal chain chr5, and 1,295,228C>T sudden change and C250T are normal chain chr5,1,295,250 C>T;

Fig. 4 is that chromatin co-immunoprecipitation (ChIP) test detects TERT expression analysis result, and wherein WT is that wild-type, C228T are normal chain chr5,1,295,228C>T sudden change and C250T are normal chain chr5,1,295,250 C>T;

Fig. 5 is that cut healing experiment detects the result of TERT promoter mutation to the invasive ability of transitional cell bladder carcinoma cell line, and wherein WT is that wild-type, C228T are normal chain chr5,1,295,228C>T sudden change and C250T are normal chain chr5,1,295,250 C>T;

Fig. 6 is the Analysis on pathologic factors result figure of TERT promoter mutation and bladder cancer patients.

embodiment:

For making technical scheme of the present invention be convenient to understand, below in conjunction with concrete test example, to the single nucleotide polymorphism sequence of TERT promotor of the present invention, the application in detecting urologic neoplasms is further described.

test example 1: by the single nucleotide polymorphism sequential detection urologic neoplasms of TERT promotor:

One, sample source and choice criteria:

With the urinary system cancer patient's of the new diagnosis of member mechanism of Chinese genitourinary system carcinoma disease genomics association (UCGC) tumor tissues, as experimental group, use patient's peripheral blood or form normal bladder tissue as a control group simultaneously.After all specimen collections by liquid nitrogen quick freezing and be stored in immediately in the environment of-80 ℃ for farther research.By two independently pathologist assess under the microscope the cancerous tissue section by hematoxylin-eosin (HE) dyeing preparation.In this research, only there is tumour cell to surpass 80% tumor tissues and be used to extract DNA and carry out order-checking subsequently.In research, always have at present 302 genito-urinary system tumour patients and participate in order-checking, comprise 216 bladder cancer patients, 11 carcinoma of renal pelvis patients, 24 renal cell carcinoma patients, 21 Patients Treated with Adrenal Neoplasms, 17 carcinoma of testis patients and 13 patients with prostate cancer.

The choice criteria of sample: 1, all patients (302 example) are in the preoperative without radiotherapy or chemotherapy; 2, patient Dou Shiyouzhong mountain tumor center makes a definite diagnosis; 3, to organize be all flesh tissue to sample, cuts in latter 30 minutes and put into RNAlater, and spend the night 4 ° of C refrigerations, thereafter-80 ° of C low-temperature storage; 4, through HE dyeing, tumour cell surpasses 80% tumor tissues; 5, Zheng Chang Alto nephridial tissue shows that in pathological examination normal uriniferous tubules and renal glomerulus and negative for tumor cells pollute; 6, the age is greater than 18 years old.

Two, main agents and instrument:

QIAamp DNA Mini Kits (test kit, Xi Erdeng, Germany); Dual 96-well GeneAmp PCR System 9700 (U.S., Life Technologies company); ABI 3730 XL sequenators (Applied biosystems); TAKARATaqTM Hot Start (test kit, Dalian precious biotechnology company limited).

Three, operation steps:

(1), according to the step of specification sheets in QIAamp DNA Mini Kits test kit, the DNA in 302 routine samples (cancer patients's tumor tissues is as experimental group, uses patient's peripheral blood or form normal bladder tissue as a control group simultaneously) is extracted:

1, cut and organize sample, determine the quantity of tissue, should be in 25mg for drawing materials of sample tissue.DNA output is relevant with size with organization type, and in general 1mg tissue can obtain the DNA of about 0.2-1.2 μ g.

2, by the tissue sample chopping (step2a) cutting down, grind (step2b):

2a. is cut into small pieces the tissue sample of approximately 25 milligrams.Put into a 1.5ml centrifuge tube, and add 180 μ l Buffer ATL.

2b. is being placed in liquid nitrogen by tissue sample, after grinding mill is thorough, is poured into 1.5ml centrifuge tube.Add 180 μ l Buffer ATL.In this process, liquid nitrogen can volatilize, but tissue can not melt.

3, add 20 μ l proteinase K, oscillation treatment 20s, 56 ℃ of water-baths are to organizing complete cracking.

Note: proteinase K must be used, because QIAGEN Protease activity in the situation that Buffer ATL exists reduces.Pyrolysis time is depending on tissue block size, and 1-3h can complete cracking conventionally.The processing of spending the night is reasonably, has no adverse effects.For guaranteeing lysis efficiency, should use shaking bath or oscillating table.

4,1.5 milliliters of microcentrifuges are centrifugal in short-term, to remove in centrifuge tube lid along liquid.

5, add 200 μ l Buffer AL, vibration 15S, 70 ℃ of water-bath 10min.1.5 milliliters of microcentrifuges are centrifugal in short-term, to remove in centrifuge tube lid along liquid.

Add after Butter AL, may produce white depositions, in 70 ℃ of water-baths, can dissolve greatly.These throw outs are for extractive process and not impact of subsequent experimental.

6, add 200 μ l ethanol (concentration is 96%-100%), vibration 15s, 1.5 milliliters of microcentrifuges are centrifugal in short-term, to remove in centrifuge tube lid along liquid.Add after ethanol, may produce white depositions.These throw outs are for extractive process and not impact of subsequent experimental.

7, careful above gained mixed solution (comprising throw out) is carefully joined in QIAamp Mini spin column, centrifugal column is put into 2ml collection tube.Cover tightly centrifuge tube lid, with the centrifugal 1min of 6000 * g (8000rpm).Centrifuge tube is taken out, put into a clean 2ml collection tube (test kit provides), abandon old collection tube and filtrate.6000 * g (8000rpm) is centrifugal is that maximum velocity centrifugation does not affect DNA output and purity in order to reduce noise.Must make all solution enter into collection tube, as not have, need again centrifugal until all solution enters collection tube with more speed.

8, carefully add 500 μ l Buffer AW1 in QIAamp Mini spin column, cover tightly centrifuge tube lid, with the centrifugal 1min of 6000 * g (8000rpm).Centrifuge tube is taken out, put into a clean 2ml collection tube (test kit provides).Abandon collection tube and worry liquid wherein.

9, carefully add 500 μ l Buffer AW2 in QIAamp Mini spin column; Cover tightly centrifuge tube lid, with the centrifugal 3min of 20000 * g (14000rpm).

10, QIAamp Mini spin column is inserted in a new 2ml collection tube (providing for oneself), abandon old collection tube and filtrate.The centrifugal 1min of full speed.This step is residual in order to prevent Buffer AW2.

11, QIAamp Mini spin column is inserted in a clean 1.5ml collection tube (providing for oneself), abandon old collection tube and filtrate.Carefully in QIAamp Mini spin column, add 200 μ l Buffer AE or distilled water.Standing 1min under room temperature, the centrifugal 1min of 6000 * g (8000rpm).

12, repeat the centrifugally operated of step 11.Collection tube being built, be stored in-20 ℃, is now the DNA of tumour and normal control in collection tube.

(2), PCR design of primers:

For TERT core promoter primers, with primer5.0, design primer:

TF: 5’-CAGCGCTGCCTGAAACTC-3’；

TR: 5’-GTCCTGCCCCTTCACCTT-3’。

(3), carry out pcr amplification: utilize the Dual 96-well GeneAmp PCR System 9700 (U.S., Life Technologies company) pcr amplification instrument, the DNA profiling obtaining from step () with primer amplification identical in step (two). according to the specification sheets of TAKARATaqTM Hot Start PCR test kit, require to amplify promotor segment:

(1), by the component preparation PCR reaction system (carrying out the preparation of reaction system on ice) in table 1.

Table 1 PCR reaction system

Figure 2013105148195100002DEST_PATH_IMAGE001

(2), in the amplification of the enterprising performing PCR of eppendof PCR thermal cycler, thermal cycle conditions is: the sensitization reaction of 94 ℃ of 5 minutes enzymes; 94 ℃ 30 seconds, 65 ℃ 30 seconds and 72 ℃ 30 seconds, totally 35 circulations; 72 ℃ 10 minutes;

(3), by the PCR product obtaining 4 ℃ of preservations.

By above-mentioned pcr amplification, obtain the PCR product of tumor group and Normal group.

(4), the PCR segment that amplifies delivers to BGI-Shenzhen, utilizes ABI 3730 XL sequenators to carry out sanger order-checking, then the peak figure obtaining is found to somatic mutation site with novosnp software analysis, sum up as shown in Fig. 1 of accompanying drawing.

(5). (the first in SEQ NO.ID 4 is chr5,1,296 to the result SEQ NO.ID 4 obtaining by above-mentioned steps, 562 position, ends are chr5,1,295,163) (in SEQ NO.ID 3, first place is chr5 with normal sequence SEQ NO.ID 3,1,296,562 position, ends are chr5,1,295,163) comparative analysis obtains: the chr5 p15 of TERT; Three places sudden change on 33 anti-chains: chr5,1,295,250 G>A (10.2%); Chr5,1,295,228 G>A (42.6%); Chr5,1,295,242-1,295,243 GG>AA (1.4%), thus obtain the chr5 p15 of TERT; Chr5 on 33,1,295,250 C>T; Chr5,1,295,228 C>T; Chr5,1,295,242-1, this three places mutantional hotspot of 295,243 CC>TT; As in SEQ NO.ID 4 the 1313rd, as shown in 1320-1321,1335.

Result:

One, the Incidence of TERT promoter gene sudden change in urogenital neoplasm:

The Incidence of TERT promoter gene sudden change in table 2 urogenital neoplasm

As shown in table 2,43% (130/302) urogenital tract tumour shows the somatic mutation of TERT promotor, and the frequency difference between dissimilar tumour of these transgenations is large (table 2) very.High-frequency sudden change occurs in two kinds and derives from the epithelial two class tumours of uropoiesis, comprises bladder cancer and carcinoma of renal pelvis.In our sample, in bladder cancer (55.6%) and carcinoma of renal pelvis (63.7%), all there is the sudden change of reverse transcriptase of telomere core promoter, but these sudden changes are comparatively rare in renal cell carcinoma (8.3%) and adrenal tumor (4.8%), also undiscovered at carcinoma of testis and prostate cancer.In our sample gene group, chr5:1295228 G>A and two mutantional hotspots of ch5:1295250 G >A (correspond to respectively chr5,1,295,228 C>T, chr5,1,295,250 C>T) especially common (shown in Fig. 1 and table 2).Chr5,1,295,228 G>A sudden changes incidence in bladder cancer and broad-mouthed receptacle for holding liquid cancer is respectively 42.6%, 45.5%.Chr5,1,295,250 G>A mutation rates are respectively 10.2%, 18.2%.In renal cell carcinoma and Patients Treated with Adrenal Neoplasm, only have chr5,1,295,228 G>A sudden changes can be observed (being respectively 8.3% and 4.8%).In addition, we also find the low frequency in other two sudden changes of bladder cancer patients, comprise that single bases G → T replaces the position chr5:1295242-1295243 (chr5 of chr5:1295228 and series connection GG > AA sudden change, 1,295,242-1,295,243 CC>TT).Two kinds of low frequency sudden changes can only be observed in 1.4% (3/216) tumour patient.This test shows that TERT promoter mutation can cause the active increase of ETRT, is the key point place that causes malignant tumour to occur.

test example 2: the external functional analysis test of TERT promoter mutation:

According to the result of the TERT promoter mutation obtaining of above-mentioned test example 1, in order to assess the impact of TERT promoter mutation on bladder cancer function, we carry out a series of external functional analysis tests:

Main agents and or instrument:; Lipofectamine 2000 (test kit, the U.S., Life Technologies company); SYBR Green (test kit, Roche Diagnostics company, the U.S.); Enzyme dyeing IP external member (Cell Signaling company); ETS1 antibody (Santa Cruz, SC-55581); Come card DMI 6000 B microscopes (come card, Wei Ci, Germany).

One, method:

Chr5 described in following method, 1,295,228 C>T and chr5,1,295,250 C>T are normal chain, and its corresponding anti-chain is chr5,1,295,228 G>A and chr5,1,295,250 G>A.

(1), by TALENs method, mutantional hotspot being introduced to urinary bladder carcinoma T24 cell line is.

Adopt TALENs (referring to Shuji Takada, Tempei Sato, Yoshiaki Ito, Satoshi Yamashita1, Tomoko Kato1, Miyuri Kawasumi, Masami Kanai-Azuma, Arisa Igarashi1, Tomomi Kato, Moe Tamano1, Hiroshi Asahara:Targeted Gene Deletion of miRNAs in Mice by TALEN System.PLOS ONE October 2013, Volume 8, Issue 10, e76004) said mutation focus is introduced and is positioned at TERT transcription initiation site upstream chr5, 1, 295, 228 C>T and chr5, 1, 295, the position of 250 C>T (chr5 on anti-chain, 1, 295, 228 G>A and chr5, 1, 295, 250 G>A), by GoldenGate method, be incorporated in T24 cell.In brief, will be for TERT promotor chr5,1,295,228 and chr5,1, it is upper that the TALENs in 295,250 sites is cloned into mammalian expression vector pCDNA3.1 (-), and use Lipofectamine 2000 (test kits according to the specification sheets of manufacturers, the U.S., Life Technologies company) transfection importing T24 clone.

(2), chromatin co-immunoprecipitation (ChIP) test

According to Shuji Takada, Tempei Sato, Yoshiaki Ito, Satoshi Yamashita1, Tomoko Kato1, Miyuri Kawasumi, Masami Kanai-Azuma, Arisa Igarashi1, Tomomi Kato, Moe Tamano1, Hiroshi Asahara:Targeted Gene Deletion of miRNAs in Mice by TALEN System.PLOS ONE October 2013, Volume 8, Issue 10, e76004. middle operational standard, use simple chip enzyme dyeing IP external member (#9003, Cell Signaling company), application ETS1 antibody (SC-55581, Santa Cruz company, ) to carrying wild-type (WT) and promoter mutation (chr5 on anti-chain, 1, 295, 228 G> A and chr5, 1, 295, 250 G> A) T24 cell is that sample carries out chip detection.

(3), TERT expression analysis

Utilize real-time quantitative PCR (RT-PCR) (referring to David Vilchez, Leah Boyer, Ianessa Morantte, Margaret Lutz, Carsten Merkwirth, Derek Joyce, Brian Spencer, Lesley Page, Eliezer Masliah, W. Travis Berggren, Fred H. Gage & Andrew Dillin:Increased proteasome activity in human embryonic stem cells is regulated by PSMD11.VOL489, NATURE, (13 SEPTEMBER 2012) 304-308.) analysis of TERT being expressed: utilize Light-Cycler 2 Auele Specific Primers pair: F, 5-caccctcgaggtgagacg-3, R, 5-gccgattgtgaacatggact-3 carries out.The transcription amplification product of TERT is detected by binding fluorescent dyes SYBR Green (test kit, Roche Diagnostics company, the U.S.) dyeing.The cycling condition of all amplified productions is as follows: initial 95 ℃ 10 minutes, ensuing 45 circulations need 94 ℃ 25 seconds, at 60 ℃ 25 seconds, and 72 ℃ 15 seconds.Under existing, the magnesium chloride of 2.5 mM carries out all circulating reactions.Specific gene product is through melting curve analysis.The normal expression of gene is by expression and following primer pair: the F of house-keeping gene GAPDH, 5-gatgatgttctggagagccc-3; R, 5-ttcgtcatgggtgtgaacc-3 completes.

(4), the expression of protein immunization imprinting technology for detection TERT

According to Ting Tan, Zhi-Qi Hu:Construction and expression of retroviral vector pLEGFP-N1-TERT in preparation of seed cells for skin tissue engineering. asian Pacific Journal of Tropical Medicine (2013) 960-963. in the total protein that extracts from T24 cell of method be dissolved in 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and be transferred to film.Sample is placed in to rabbit against human T ERT monoclonal antibody and hatches at 37 ℃ 2 hours, be then stored in incubated overnight at 4 ℃.Under goat anti-rabbit igg room temperature by horseradish peroxidase-labeled, hatch after 1 hour, lower 2 minutes samples of dark condition will be dyeed by chromogenic substrate again.The internal control effect that wherein beta-actin is served as.

(5), cut healing experiment

According to Imad Al Ghouleh, Andr é s Rodr í guez, Patrick J. Pagano and G á bor Cs á nyi:Proteomic Analysis Identifies an NADPH Oxidase 1 (Nox1)-Mediated Role for Actin-Related Protein 2/3Complex Subunit 2 (ARPC2) in Promoting Smooth Muscle Cell Migration. int. J. Mol. Sci.2013, 14, 20220-20235; Doi:10.3390/ijms141020220. the method in is inoculated in the T24 cell that has the promotor of wild-type and saltant type in 6 orifice plates, reaches 80% left and right and converges.First, cell is by as for containing in the substratum of 2% foetal calf serum (FCS) hungry 24 hours.Carry out subsequently cut operation, then use the white point of 10 μ L to cure for each cut.Then, cell is taken pictures with come card DMI 6000 B microscopes (come card, Wei Ci, Germany) after rinsing with PBS.The cell in injured region was hatched after 24 hours, then carried out once photo taking.

Two, result: (the external functional analysis of TERT promoter mutation)

In order to assess the potential function consequence of TERT promoter mutation, we by TALENs by chr5,1,295,228G> A and chr5,1,295,250G> A sudden change imports urinary bladder carcinoma T24 cell line strain, then observes the changes of function that these sudden changes cause.The result of RT-PCR and immunoblotting assay (seeing Fig. 2 and Fig. 3) shows, compare WT cell strain, have chr5,1,295,228 G> A or chr5,1, the transitional cell bladder carcinoma cell line TERT expression level of 295,250 G> A mutation alleles is showing unrestricted.This result shows, chr5, and 1,295,228 G> A and chr5,1,295,250 G> A sudden changes may be conducive to the transcription factor combination of TERT promotor, thereby strengthen the expression of TERT.Due to determining of single base in tumour and series connection sudden change, we guess that TERT promoter mutation produces the transcription factor Ets1 of the binding motif of sudden change, and we use anti-Ets1 antibody to carry out chromatin Immunoprecipitation and detect the combination whether sudden change can increase transcription factor and TERT promotor.Compare with non-specific IgG control antibodies, in the T24 cell of sudden change but not in WT T24 cell, can be observed TERT start with ETS1 antibody showing enrichment (seeing Fig. 4).This result of study is confirmed, chr5, and 1,295,228G> A and chr5,1,295,250G> A sudden change has strengthened the combination between TERT promotor and transcription factor ETS1.

For whether research TERT promoter mutation can affect the invasive ability of transitional cell bladder carcinoma cell line, we have carried out wound healing experiment, with the cell viability of the cell of the WT that checks and mutation T 24.After 24h, we observe and compare wild-type to group, chr5, and 1,295,228 G> A and chr5,1,295,250 G> A mutant cell experimental group are all showing promotion cut healing (seeing Fig. 5).This result proves, chr5 in TERT promotor, and 1,295,228G> A and chr5,1,295,250 G> A sudden changes all can improve the invasive ability of transitional cell bladder carcinoma cell line.

test example 3: tERT sudden change and bladder cancer (216 examples in test example 1: concrete information summary is as table 3) clinical characteristic correlation analysis:

The Analysis on pathologic factors of table 3 bladder cancer patients

Patient characteristics (Clinical features)	Sudden change (# of samples with mutations)	Not mutated (# of samples without mutations)	P value ( P value)
				Show shallow /Infiltrating
Show shallow	39	49	0.0059
				Infiltrating	81	47
Invasive depth-classification
				Ta-1	56	58	0.0443
T2-4	64	38
				Sex
Man	107	77	0.0655
				Female	13	19
Age
				10-49	15	25	0.0109
50-89	105	71

The Analysis on pathologic factors of TERT promoter mutation and bladder cancer patients: shown in Fig. 6 and table 3, clinically, there is two different bladder cancer (TCC) group, case up to 70% is non-flesh layer invasive bladder cancer (the NMI TCCS on top layer, Ta and T1 phase), this class patient is easy to recurrence, but general entail dangers to life not; And be invasive bladder cancer (MI TCCS, the T2 – T4 phase) up to 30% case, this class patient is owing to existing distant metastasis to have higher mortality ratio.In our queue of TCC, we find that TERT promoter mutation is all relevant with transitional cell carcinoma invasive ability.In MI-TCC hypotype TERT promoter mutation rate (81/128,63.3 %), be significantly higher than NMI-TCC hypotype (39/88,44.3 %; Application Pearson chi square test obtains P=0.0059).This result shows that TERT promoter mutation may improve the invasiveness of TCC tumour cell, promotes TCC to occur and progress.Therefore, our data presentation: than TERT promotor in low level hypotype (49.1%, Ta and T1 56/114) mutation frequency, mutation frequency in high-level TCC hypotype (62.7%, T2-4 64/102) higher (P < 0.05).This result shows that TERT promoter mutation may be present in the late incident that development occurs TCC, and for the prediction of distant metastasis risk.We also find that male patient's TERT promoter mutation frequency (58%, 107/184) is higher than female patient (41%, 13/32), but difference significantly (is not applied Pearson's chi square test and obtained P=0.0655).In addition the frequency (59.7%, 105/176) that, we find the TERT promoter mutation that 50 years old age of patient is above is apparently higher than the right side of fifty (37.5%, 15/40) (application Pearson chi square test obtains P=0.0109).This result shows, the elderly TERT promoter mutation more may promote generation, the development of TCC.In the present invention, by the single nucleotide polymorphism sequential detection urologic neoplasms of TERT promotor, screened the sudden change of the TERT promotor of 302 urogenital tumours, and several high frequency mutantional hotspots have been determined, by test example 2, carried out external functional analysis, show and carried out relevant external functional analysis and tested to confirm: the sudden change of these promotors can improve ETRT activity, it is the key point place that causes malignant tumour to occur, also analyze applying clinical pathological factor simultaneously and assessed TERT gene as the effect of the biomarker of early-stage cancer detection and prognosis, these sudden changes have great importance to the diagnosis and prognosis of urogenital system malignant tumour.

The above, it is only preferred embodiment of the present invention, not the present invention is done to any formal and substantial restriction, all those skilled in the art, do not departing within the scope of technical solution of the present invention, when utilizing disclosed above technology contents, and the equivalent variations of a little change of making, modification and differentiation is equivalent embodiment of the present invention; Meanwhile, the change of any equivalent variations that all foundations essence technology of the present invention is done above embodiment, modification and differentiation, all still belong in the scope of technical scheme of the present invention.

SEQUENCE LISTING

<110> Shenzhen City Second People's Hospital

The application of the single nucleotide polymorphism sequence of <120> TERT promotor in detecting urologic neoplasms

<130>

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 18

<212> DNA

<213> primer pair P middle and upper reaches primer sequence TF

<220>

<221> misc_feature

<222> (1)..(18)

<400> 1

cagcgctgcc tgaaactc 18

<210> 2

<211> 18

<212> DNA

<213> primer pair P middle and upper reaches primer sequence TP

<220>

<221> misc_feature

<222> (1)..(18)

<400> 2

gtcctgcccc ttcacctt 18

<210> 3

<211> 1400

<212> DNA

<213> TERT normal sequence

<400> 3

gtggcagaga caattcacaa acacagccct ttaaaaaggc ttagggatca ctaaggggat 60

ttctagaaga gcgacctgta atcctaagta tttacaagac gaggctaacc tccagcgagc 120

gtgacagccc agggagggtg cgaggcctgt tcaaatgcta gctccataaa taaagcaatt 180

tcctccggca gtttctgaaa gtaggaaagg ttacatttaa ggttgcgttt gttagcattt 240

cagtgtttgc cgacctcagc tacagcatcc ctgcaaggcc tcgggagacc cagaagtttc 300

tcgcccctta gatccaaact tgagcaaccc ggagtctgga ttcctgggaa gtcctcagct 360

gtcctgcggt tgtgccgggg ccccaggtct ggaggggacc agtggccgtg tggcttctac 420

tgctgggctg gaagtcgggc ctcctagctc tgcagtccga ggcttggagc caggtgcctg 480

gaccccgagg ttgccctcca ccctgtgcgg gcgggatgtg accagatgtt ggcctcatct 540

gccagacaga gtgccggggc ccagggtcaa ggccgttgtg gctggtgtga ggcgcccggt 600

gcgcggccag caggagcgcc tggctccatt tcccaccctt tctcgacggg accgccccgg 660

tgggtgatta acagatttgg ggtggtttgc tcatggtggg gacccctcgc cgcctgagaa 720

cctgcaaaga gaaatgacgg gcctgtgtca aggagcccaa gtcgcgggga agtgttgcag 780

ggaggcactc cgggaggtcc cgcgtgcccg tccagggagc aatgcgtcct cgggttcgtc 840

cccagccgcg tctacgcgcc tccgtcctcc ccttcacgtc cggcattcgt ggtgcccgga 900

gcccgacgcc ccgcgtccgg acctggaggc agccctgggt ctccggatca ggccagcggc 960

caaagggtcg ccgcacgcac ctgttcccag ggcctccaca tcatggcccc tccctcgggt 1020

taccccacag cctaggccga ttcgacctct ctccgctggg gccctcgctg gcgtccctgc 1080

accctgggag cgcgagcggc gcgcgggcgg ggaagcgcgg cccagacccc cgggtccgcc 1140

cggagcagct gcgctgtcgg ggccaggccg ggctcccagt ggattcgcgg gcacagacgc 1200

ccaggaccgc gcttcccacg tggcggaggg actggggacc cgggcacccg tcctgcccct 1260

tcaccttcca gctccgcctc ctccgcgcgg accccgcccc gtcccgaccc ctcccgggtc 1320

cccggcccag ccccctccgg gccctcccag cccctcccct tcctttccgc ggccccgccc 1380

tctcctcgcg gcgcgagttt 1400

<210> 4

<211> 1400

<212> DNA

<213> TERT mutant nucleotide sequence

<400> 4

gtggcagaga caattcacaa acacagccct ttaaaaaggc ttagggatca ctaaggggat 60

ttctagaaga gcgacctgta atcctaagta tttacaagac gaggctaacc tccagcgagc 120

gtgacagccc agggagggtg cgaggcctgt tcaaatgcta gctccataaa taaagcaatt 180

tcctccggca gtttctgaaa gtaggaaagg ttacatttaa ggttgcgttt gttagcattt 240

cagtgtttgc cgacctcagc tacagcatcc ctgcaaggcc tcgggagacc cagaagtttc 300

tcgcccctta gatccaaact tgagcaaccc ggagtctgga ttcctgggaa gtcctcagct 360

gtcctgcggt tgtgccgggg ccccaggtct ggaggggacc agtggccgtg tggcttctac 420

tgctgggctg gaagtcgggc ctcctagctc tgcagtccga ggcttggagc caggtgcctg 480

gaccccgagg ttgccctcca ccctgtgcgg gcgggatgtg accagatgtt ggcctcatct 540

gccagacaga gtgccggggc ccagggtcaa ggccgttgtg gctggtgtga ggcgcccggt 600

gcgcggccag caggagcgcc tggctccatt tcccaccctt tctcgacggg accgccccgg 660

tgggtgatta acagatttgg ggtggtttgc tcatggtggg gacccctcgc cgcctgagaa 720

cctgcaaaga gaaatgacgg gcctgtgtca aggagcccaa gtcgcgggga agtgttgcag 780

ggaggcactc cgggaggtcc cgcgtgcccg tccagggagc aatgcgtcct cgggttcgtc 840

cccagccgcg tctacgcgcc tccgtcctcc ccttcacgtc cggcattcgt ggtgcccgga 900

gcccgacgcc ccgcgtccgg acctggaggc agccctgggt ctccggatca ggccagcggc 960

caaagggtcg ccgcacgcac ctgttcccag ggcctccaca tcatggcccc tccctcgggt 1020

taccccacag cctaggccga ttcgacctct ctccgctggg gccctcgctg gcgtccctgc 1080

accctgggag cgcgagcggc gcgcgggcgg ggaagcgcgg cccagacccc cgggtccgcc 1140

cggagcagct gcgctgtcgg ggccaggccg ggctcccagt ggattcgcgg gcacagacgc 1200

ccaggaccgc gcttcccacg tggcggaggg actggggacc cgggcacccg tcctgcccct 1260

tcaccttcca gctccgcctc ctccgcgcgg accccgcccc gtcccgaccc cttccgggtt 1320

tccggcccag ccccttccgg gccctcccag cccctcccct tcctttccgc ggccccgccc 1380

tctcctcgcg gcgcgagttt 1400

Claims

The application of the single nucleotide polymorphism sequence of 1.TERT promotor in detecting urologic neoplasms, the single nucleotide polymorphism sequence of described TERT promotor is:

TERT promotor chr5,1,295,228 is the nucleotide polymorphisms sequence of C or T;

TERT promotor chr5,1,295,242-1,295,243 is the nucleotide polymorphisms sequence of CC or TT;

TERT promotor chr5,1,295,250 is the nucleotide polymorphisms sequence of C or T.
2. the application of the single nucleotide polymorphism sequence of TERT promotor according to claim 1 in detecting urologic neoplasms, is characterized in that: described urologic neoplasms is transitional cell carcinoma of bladder or upper Urothelium carcinoma.
3. the application of the single nucleotide polymorphism sequence of TERT promotor according to claim 1 and 2 in detecting urologic neoplasms, is characterized in that, described application comprises the steps:

(1), from sample urinary system tissue, extract DNA;

(2), the testing gene group DNA that comprises TERT promoter gene of take is template, take primer pair P as primer, pcr amplification TERT promoter gene, described primer pair P is:

TF：5’-CAGCGCTGCCTGAAACTC-3’，

TR：5’-GTCCTGCCCCTTCACCTT-3’；

After pcr amplification completes, PCR product is carried out to sanger order-checking, while containing the single nucleotide polymorphism sequence of TERT promotor in sample, this sample is transitional cell carcinoma of bladder or upper Urothelium carcinoma sample.
4. the application of the single nucleotide polymorphism sequence of TERT promotor according to claim 3 in detecting urologic neoplasms, it is characterized in that, described PCR reaction system volume is 20 μ l, contains dNTP 2 μ l, 10 * PCR Buffer, 2 μ l, TF (10 μ M) 1 μ l, TR (10 μ M) 1 μ l, template DNA 1 μ l, dH ₂o 12.5 μ l and TaKaRa Taq HS 0.5 μ l.
5. the application of the single nucleotide polymorphism sequence of TERT promotor according to claim 3 in detecting urologic neoplasms, it is characterized in that, described pcr amplification reaction program is: 93 ℃ of 2 minutes denaturations, then by 93 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, totally 40 do circulation, last 72 ℃ are extended for 7 minutes.