Identification Of Prostate Cancer Risk SNPs In Northern Chinese Men And The Functional Study Of GPRC6A Gene

Prostate cancer is a complex polygenic inheritance disease and is threatening the healthy of the men severely. The established risk factors are age, ethnicity, and a positive family history of disease. It has been reported that there were approximately50prostate cancer susceptibility loci identified in the western and Japanese populations, however, the distribution of these loci and the association with prostate cancer in northern Chinese population is unknown, and the function of these prostate cancer risk loci has not been studied efficiently. As consequence, the development of some molecular approach that can be assumed as noninvasion diagnosis at early stage and prognosis will be limited. So earring out the identification, replication and confirmation of tumor markers will not only enhance the understanding of the complex genetic effect on prostate carcinogenesis but also contribute to the searching for diagnostic markers of prostate cancer, to service patients better. Some genetic markers are being as one of cancer biomarkers. It is nessary to study prostate cancer and search for the specific tumor markers.Objective Identify the genetic prostate cancer markers and explore their function. Part1:To confirm the risk variants in northern Chinese population. Part2:To explore the effect of GPRC6A gene in the metastasis of prostate cancer cells. Part3:Preparation and identification the monoclonal antibody of the expression product from TMPRSS2-ERG fusion.MethodsPart1(Identification of prostate cancer susceptibility genes):1) We selected the reported36prostate cancer susceptibility SNPs from several GWAS on prostate cancer in some countries, and designed to genotyping in northern Chinese men including286cases and288controls. We explored the association between clinical covariates (age at diagnosis, prostate-specific antigen levels, Gleason score, and tumor stage) and prostate cancer risk SNPs found in our study to infer their impact on aggressive prostate cancer. We also evaluate the cumulative effect among the risk SNPs.2) We selected the tag SNPs from the genes anchored prostate cancer risk loci by using Haploview software combined with the biologic database, and performed association study between these SNPs and prostate cancer in286cases and630control subjects.3) Haplotype-based associations were analyzed between prostate cancer cases and control individuals.Part2(Functional study of prostate cancer susceptibility gene):1) The expressions of GPRC6A protein on normal prostate epithelial cell RWPE-1, prostate cancer epithelial cells including PC3, LNcap and Vcap cells were examined by using western blot method.2) PC3cells transfected transiently with GPRC6A siRNA or universal siRNA control were used to determine metastasis behavior.3) Scratch wound healing assay and transwell migration assay were used to evaluate the migration ability of PC3cells and transwell invasion assay was used to estimate the invasion ability of PC3cells. The potential mechanism that affected the metastasis efficiency caused by GPRC6A gene was explored by analyzing the MMPs protein levels.Part3(Application study of TMPRSS2-ERG fusion):1) Understanding the biologic function of the fusion TMPRSS2-ERG gene by analyzing the reported studies and determined the expression product.2) Prepare the monoclonal antibody and identify its utilization in prostate cancer cells.ResultsPart1(Identification of prostate cancer susceptibility genes):1) Six of the36SNPs were associated with prostate cancer risk, including4SNPs in8q24,2SNPs at6chromosomes, respectively.2) The risk alleles of these risk SNPs excepting rs339331were associated with at least one clinical covariates compared with controls.3) The cumulative effect of these6SNPs indicated that compared to men who did not have any of these risk variants, men who carried any combination of1,2or^3risk genotypes have a gradually increased prostate cancer risk and the dramatic OR value (OR=1.79-4.14, p=0.042-2.22×10-6).4) Nine risk SNPs at GPRC6A and RFX6genes and one SNP at FOXP4gene were associated with prostate cancer.5) Some haplotypes in8q24, GPRC6A, RFX6, and FOXP4genes were associated with prostate cancer.Part2(Functional study of prostate cancer susceptibility gene):1) Expression of GPRC6A gene was higher in PC3and Vcap cells those derived from bone than in normal prostate epithelial cell RWPE-1and the LNcap cell that derived from the supraclavicular lymph node.2) Both of the migration and the invasion abilities in GPRC6A knock-down cells were lower compared with siRNA control cells.3) Expression trend of MMP2and MMP4was same to GPRC6A.Part3(Application study of TMPRSS2-ERG fusion):1) The prepared monoclonal antibody of TMPRSS2-ERG is consistent with the commercial ERG antibody.2) The TMPRSS2-ERG antibody can be used in western blot and immunohistochemisty study.Conclusion1. Six prostate cancer susceptibility SNPs are confirmed and10prostate cancer susceptibility variations at GPRC6A, RFX6and FOXP4genes are identified in northern Chinese men.2. GPRC6A gene expression is associated with the migration and the invasion abilities of prostate cancer cells, which may be mediated by MMP2and MMP9proteins.3. The TMPRSS2-ERG monoclonal antibody has potential ability to be applied in the testing of prostate cancer.