The Mechanism Study Of UGT1A3 Low Frequency Variant Associated With Bladder Cancer Risk Through Target Region Sequencing

Bladder cancer is the most common malignant cancer in the urinary system,which imposed a heavy burden on social development.Previous epidemiological studies have shown that smoking and occupational exposure are the major risk factors of bladder cancer.Expect for environmental risk factors,genetic variations play a key role in the susceptibility of bladder cancer.Among them,single nucleotide polymorphism(SNP)was widely investigated.With the development of high-through genotyping technology,the study strategy of SNP stepped from candidate gene to candidate pathway and genome-wide association study(GWAS).It is generally agreed upon that GWAS had made great contributions to reveal a variety of disease-related susceptibility loci.Up to now,GWASs have identified multiple region including seventeen susceptibility loci of bladder cancer.However,the majority of all identified SNPs are common variants with minor allele frequency more than 0.05 and had moderate effect with odds ratio smaller than 1.5.Some studies have demonstrated that variants identified explain only less than 10%of the inherited risk,which indicate that there are missing heritability of bladder cancer need to be found.The common disease-rare variant hypothesis suggests that the additional heritability can be explained by low frequency and rare variants with stronger effect.In the post-GWAS era,researchers conducted genotype imputation to integrate the existing GWAS data from different platforms into the same genotype density.Then,meta-analysis of the imputation data was applied to identify novel common variants and disease-related rare susceptibility loci.However,the quality of imputation was influenced by density of GWAS data and imputation softwares,which may result in low quality even missing genotype of several loci.With the rapid development of sequencing technology,next-generation sequencing has been widely applied in genome study,in which target region sequencing is the most cost-effective strategy.Considering the important role of GWAS-identified susceptibility regions in the development of cancer,several studies conducted target region sequencing of the key genes in the region,and found that novel low frequency variants were related to the disease risk.So far,little is known of the associations between low frequency variants in the GWAS-identified regions and bladder cancer risk.This study applied target region sequencing to detect bladder cancer-related low frequency variants in the GWAS-identify regions and two-stage case-control studies were conducted to further confirm the associations.Moreover,we applied molecular biology experiments to explore the underlying mechanism of susceptibility genes and low frequency variants involved in the carcinogenesis of bladder cancer.Our study may provide beneficial information for etiology and screening of high-risk population of bladder cancer.Part 1 Target region sequencing-based association analysis of low frequency variants and the risk of bladder cancerBackgroundGWASs have identified seventeen regions that contain bladder cancer susceptibility loci:8q24.3(PSCA),2q37.1(UGT1A),5p15.33(TERT-CLPTM1L),3q28(TP63)and so on.However,the identified SNPs could explain the few genetic effect of bladder cancer,and the biological function of risk loci are largely unexplored.Emerging evidence has shown that low frequency and rare variants in the GWAS-identified susceptibility regions may explain some of the missing heritability.Target region sequencing is a cost-effective and efficient strategy to explore unrevealed low frequency and rare variants that are related to complex disease in large cohorts.There is no literature about the associations between low frequency and rare variants in GWAS-identified susceptibility region and the risk of bladder cancer.MethodsWe calculated the recombination hotspots and screening the candidate genes in the susceptibility region.Target region sequencing in 113 cases and 118 cancer-free controls was performed to evaluate the bladder cancer associated risk loci and genes.Gene-base analysis and single-variant logistic regression analysis were used to assess the associations between low frequency variants and the bladder cancer risk.In addition,the association analyses were also applied in another two independent case-control studies(2,237 cases and 2,603 controls)for validation.ResultsThe gene-base analysis results showed that UGT1A3 gene was prominently related to the susceptibility of bladder cancer in additive and dominant models.Moreover,the results of multiple logistic regression analysis indicated that low frequency variant rs28898617 A>G,located in the first exon of UGT1A3,was significantly associated with increased risk of bladder cancer.Further two-stage case-control studies were conducted and significant associations between rs28898617 polymorphism and susceptibility of bladder cancer were observed in additive,dominant and co-dominant models.Besides,the combined results of target region sequencing and two-stage validation studies demonstrated that individuals carrying rs28898617 AG/GG genotype confer a 1.46-fold increased bladder cancer risk compared with those with AA genotype(OR=1.46,95%CI=1.18-1.79).Additionally,stratification analysis showed that genetic variation rs28898617 G allele had remarkable associations with increased the risk of bladder cancer in the subgroups of males and smokers.ConclusionOur study focused on the GWAS-identified susceptibility region of bladder cancer,firstly conducted target region sequencing study and revealed a significant association between rs28898617,a low frequency variant located in UGT1A3,and bladder cancer risk.These results suggest that UGT1A3 and its low frequency variant play a key role in the genetic susceptibility to bladder cancer.Part 2 The mechanism study of UGT1A3 and its susceptibility loci involved in the carcinogenesis of bladder cancerBackgroundMounting evidence shows that smoking and occupational exposure can expose individuals to environmental carcinogens,in which aromatic amine and polycyclic aromatic hydrocarbons can induce DNA damage and genome instability,thus involved in the carcinogenesis of cancer.It has been established that genetic variants,environmental factors and their interactions jointly contributed to bladder cancer.Intriguingly,genetic variants in detoxification genes may determine difference of the toxic metabolic capacity between individuals,which influenced the susceptibility to cancers.As an important member of Phase II enzymes,UGTs(UDP-glucuronosyltransferases)are mainly responsible for glucuronidation to excrete endogenous and exogenous substances.Our previous study results indicated that UGT1A3 rs28898617 was significantly related to bladder cancer risk.Therefore,further functional study was conducted to explore the mechanism of UAT1A3 and SNP rs28898617 in the carcinogenesis of bladder cancer.MethodsIn this study,we used the luciferase assay and expression quantitative trait loci(eQTL)analysis to evaluate the effect of risk loci on transcriptional activity and the expression level of susceptibility gene.Besides,a series of molecular biology experiments were performed to demonstrate the effect of susceptibility gene on the maligant phenotype of bladder cancer.Furthermore,we investigated underlying regulation mechanism of UGT1A3 implicated in bladder cancer.ResultsThe luciferase assay and eQTL results indicated that rs28898617 G alle:le significantly enhanced the transcriptional activity and the expression of UGT1A3,compared with A allele.Additionally,knockdown of UGT1A3 in bladder cancer cell lines inhibited cell proliferation,invasion and migration,whereas overexpression of UGTIA3 could abolish this effect.In the further study,we found that androgen receptor(AR)expression was positively associated with UGT1A3 mRNA expression in bladder cancer tissues.Besides,electrophoretic mobility shift assay(EMSA)revealed that rs28898617 G allele strengthen binding ability of transcription factors.We also found that overexpression or knockdown of AR in bladder cancer cell lines substantially increased or decreased the expression of UGT1A3.Furthermore,overexpression of UGT1A3 could abolish the effect of AR on cell phenotypes.These results suggest that rs28898617 G allele increased the expression level of UGT1A3 mediated by promoting binding efficiency of AR.ConclusionTaken together,the present study demonstrated that rs28898617 A>G regulated the expression of UGT1A3 through altering the binding ability of AR and thus involved in susceptibility to bladder cancer.This study investigated the molecular mechanism of UGT1A3 and its susceptibility loci implicated in development of bladder cancer,which may provide new insights into the mechanism study of low frequency variants.