Relationship Between CAG Repeat Polymorphism Of Androgen Receptor Gene And Androgen Induced TMPRSS2:ERG Fusion

Prostate cancer (PCa) is the most common male malignancy and thesecond leading cause of cancer mortality among men in Western countries.However, there is significant disparity between the incidence and mortalityof the disease among different countries and races. There are evidences toshow that PCa development is due to multiple factors, such asenvironmental exposure, diet and genetic variation. However, althoughAsian immigrants in North America and Europe have a higher incidence ofPCa than residents in Asia, it is still lower than white and black men inthose regions. Immigrant studies in the US also showed that, even underthe same environmental conditions and medical care system, there weresignificant differences in mobility and mortality of PCa between white andAfrican American menThe growth of normal prostate epithelialal cells or PCa cells depends onandrogen. AR gene is located at Xq11.2-q12, the open reading frame isseparated over eight exons that encode for AR. The amino-terminal transcriptional activation domain, encoded by exon one, includes a highfrequency polymorphic repeats, CAG. AR expression level and functionwere found to have an inverse association with the length of CAG in vitrostudies, The length of CAG repeats ranges from8to35repeats in thenormal population. Hispanic men have been reported to have the longestaverage CAG repeat length (23-25), the Chinese population have longerCAG repeats (average between22-23) than that of the Caucasianpopulation (average between21-22), and the black population have theshortest average CAG repeats (average between19-20). Therefore theshorter CAG repeat was supposed to be risk for prostate cancer, althoughclinic research results are inconclusion.On the other hand, androgens have also been implicated in theoccurrence of the TMPRRS:ERG fusion gene. This fusion gene has beenfound at different frequencies between populations, occurring in50%ofPCa samples from Western men, in comparison to around10%in Chinesemen. This fusion gene can be induced in the PCa cell line LNCaPfollowing treatment for24h with DHT and in non-cancer cell line PNT1Aand PNT2following long term exposure to DHT. Therefore, androgenlevels may be an important factor in PCa risk.My project was designed to study the relationship between CAG repeatpolymorphism and amdrogen-induced TMPRSS2:ERG fusion, then todiscover the whether CAG repeat polymorphism change the prostate cancer risk in different population. My project includes the following three parts.Part1Extract DNA from prostate cancer sample and measure CAGrepeat polymorphism of the androgen receptor geneObjectiveTo explore the relationship between CAG repeat of androgen receptorand TMPRSS2:ERG fusion using DNA extracted from paraffin-embeddedsamples.MethodsExtract DNA from126paraffin-embedded prostate cancer sampleswhose TMPRSS2:ERG fusion status has been previously derermined. PCRamplify CAG repeat fragment by primer5’-ACCCAGAGGCCGCGAGCGCA and5’-TTGCTGTTCCTCATCCAGGA, then send the PCR products tosequence.Results17CAG repeat and24CAG repeat had different rate of TMPRSS2:ERGfusion, fusion positive/negative is7/3and2/12in17CAG and24CAGrespectively.ConclusionIt was supposed17CAG increased the TMPRSS2:ERG fusion rate, and24CAG decreased the TMPRSS2:ERG fusion rate. Part2Construct plasmid and DU145prostate cancer cell lines withdifferent CAG polymorphism androgen receptor geneObjectiveBased on the results of part1, I planed to construct plasmid and DU145prostate cancer cell lines with different CAG polymorphism androgenreceptor geneMethodsUse pRR-AR-5Z as template and the following primers5’-ACGGATGCTAGCATGGAAGTTCAATTGGGTTTGand5’-TTGACTTCTAGATCACTGGGTGTGGAAATAGATG, I madeandrogen receptor cDNA by proofread PCR. AR cDNA was cloned intopCR2.1TOPO T vector, transfected into competent cells, plasmid DNAextracted and then sequenced to confirm the correct sequence. Usinggenome DNA of patients with15CAG,17CAG and24CAG repeatpolymorphism respectively as template and the primers of5’-ACGGATGCTAGCATGGAAGTTCAATTGGGTTTG and5’-GCTGCTTAAGCCGGGGAAAGTGGGGCC, I PCRed CAG repeatDNA fragments. To clone CAG fragment into pCR2.1TOPO, I cut T vectorcarried CAG fragment and pcDNA3.1+carried AR by incision enzymeNheI and Aflii and then ligased the DNA fragments.DU145cells were transfected with pcDNA3.1+carried AR by lipofectamine2000and transfected DU145cells were select by G418. TheAR protein expression was measure by western blot and AR mRNA levelby realtime PCR.ResultsI constructed pcDNA3.1+plasmid carried AR with CAG polymorphism,abd transfected it into DU145cells to get stable transfected cellssuccessfully. Compared with DU145, the AR mRNA expression is2896.30,1.45,944.45,668.60,727.43in VCaP, empty plasmid transfect DU145,15CAG DU145,17CAG DU145and24CAG DU145respectively, butwestenblot can’t check the AR protein expression in transfect cells.ConclusionI have successfully constructed prostate cancer cell line DU145withCAG polymorphism AR gene by molecular cloning and cell biologicaltechnology.Part3Explore the relationship between CAG repeat polymorphism ofAR and TMPRSS2:ERG fusion by FISHObjectiveTo detect colocalization of TMPRSS2and ERG in different CAGrepeat polymorphism DU145by FISH MethodsI Cultured cell with androgen free medium for24h before experiment,treated cell3h for100nM DHT, then collected and fixed cells. cells werehybridized with RP11-35C4(TMPRSS2red) and RP11-476D17(ERGgreen) probe and counterstained with20ul DAPI to check colocalizationrate by fluorescence microscope.ResultsBefore treatment and after treatment with DHT, the rate ofcolocalization is6.95%and6.33%in untransfect DU145，6.81%and8.09%in15CAG transfected DU145，6.83%and10.92%in17CAGtransfected DU145，7.21%and9.23%in24CAG transfected DU145.ConclusionDU145cells with AR have higher TMPRSS2:ERG colocalization ratethan DU145cells without AR.17CAG repeat polymorphism cellsindecued highest colocalization percent compared with15CAG and24CAG polymorphism.