The Study Of Association Between The Genetic Variants At Chromosome3q28and Risk Of Bladder Cancer

Bladder cancer, only secondary to the prostate cancer, ranks the second amongthe most common urinary system tumors. In China, the morbidity of bladder cancerhave been increasing rapidly in last two decades, approximately70%of patientspresent as non–muscle-invasive at first diagnosis. Both genetic and environmentalfactors contribute to the pathogenesis of bladder cancer. It has been established thatcigarette smoking and occupational exposure are the two most important risk factorsfor bladder cancer. Epidemiological studies have indicated that there is aninter-individual variation in genetic susceptibility to bladder cancer under the similarexposure. Differences between genetic backgrounds are mainly in the generalpopulation for differences in genome sequences, including single nucleotidepolymorphisms (SNPs).Over the last several years, as a powerful method to investigate the geneticdeterminants of complex diseases, genome-wide association study (GWAS) hassuccessfully identified thousands of genetic markers that are related to thesusceptibility of diseases. Since the first GWAS identified two new loci containingvariants on chromosome3q28and8q24that confered bladder cancer risk in2008,there had been several susceptibility loci harboring common variants associated withan increased risk of bladder cancer till now. Therefore, we hypothesized that thegenetic variants on GWAS previously reported SNPs also play an important role inbladder cancer susceptibility in a Chinese population, and that the causal loci on thoseareas may predispose to the bladder cancer. To test these hypotheses, we conducted an evaluation of promising associationsfor bladder cancer in a two-stage case-control study. According to the results fromGWAS,“fine mapping” approach was conducted to select SNPs. The results of ourstudy will be helpful for determining the genetic factors of bladder cancer in aChinese population and elucidating the susceptibility mechanism in bladder cancer.Part I “Fine Mapping” study of genetic variants atchromosome3q28with susceptibility of bladder cancerHuman chromosome3q28region contains two genes, TP63and LEPREL1.Accumulating GWAS reports support a role of3q28region gene polymorphisms inbladder cancer risk. However, most of these GWAS are mainly focus on theCaucasian population. Since the functional relevance for genetic variants at this locusis still unclear, it is critical to systematically assess the relationship of genetic variantsin this region with the risk of bladder cancer in Chinese population.To test this hypothesis, a two-stage case-control study was conducted in Hanethnic populations derived from Jiangsu, China. The first-stage, consisting of540cases and555controls, was to discover novel risk variants from41SNPs selectedaccording to linkage disequilibrium in a Chinese population by using a fine mappingapproach. The results showed that rs35592567TT homozygous can significantlydecrease the risk of bladder cancer [adjusted odds ratio (OR)=0.35,95%confidenceinterval (CI)=0.17-0.71), rs4687112GG homozygous also significantly decreasedbladder cancer risk (adjusted OR=0.64,95%CI=0.45-0.91). Then, we validated ourresult in an independent case-control study including840bladder cancers and1044controls. The results were confirmed, rs35592567T allele decreased the risk ofbladder cancer in additive model (adjusted OR=0.81,95%CI=0.68-0.96).Compared with the TT genotype, rs4687112TG genotype was associated withdecreased risk of bladder cancer (adjusted OR=0.77,95%CI=0.60-0.99). The combined logistic regression analysis of two stage samples indicated that rs35592567T allele decreased the risk of bladder cancer by21%(Additive model: adjusted OR=0.79,95%CI=0.69-0.90). In additive model, individuals with G allele exhibited14%decreased risk of bladder cancer (adjusted OR=0.86,95%CI=0.77-0.95).In summary, these findings suggest that genetic variants of the3q28region maycontribute to bladder cancer susceptibility and rs35592567and rs4687112may berelated to bladder cancer risk in Chinese population.Part II Functional study of genetic variants at chromosome3q28with susceptibility of bladder cancerThe TP63gene, located on chromosome3q28, is a member of the p53genefamily. TP63encodes multiple isoforms that can be placed in two categories:isoforms with the N-terminal transactivation (TA) domain, which are known as theTA isoforms; and isoforms that lack the TA domain, which are known as the ΔNisoforms. Recent study has reported that TAp63could suppress metastasis bytranscriptionally activating metastasis suppressor genes or microRNAs, such asDICER1, mir-130b and BHLHE41.We found that SNP (rs35592567) located in3’-UTR of TP63was associatedwith a significantly decreased bladder cancer risk. Furthermore, the bioinformaticsprediction suggested that this SNP was located at the binding sites of miR-140-5p in3’-UTR of TP63. To evaluate whether rs35592567variant might affect the bindingsite of miRNAs, we used luciferase reporter palsmids of TP633’-UTR (containingrs35592567C or T allele) that were cotransfected with miR-140-5p mimics in T24,EJ and293A cells. The results of vitro experiment showed the variant T allele ofrs35592567decreased the binding capability of miRNAs, which might lead to theup-expression of TP63. Additional experiments with tumor tissues revealed that thelevels of TP63mRNA and protein expression in bladder cancer tissues were significantly increased compared with paired adjacent normal tissues.Overall, these findings indicate that rs35592567located at miR-140-5p bindingsite may alter expression of TP63though breaking miRNA binding sites andcontribute to the suppression of bladder cancer.