The Study On Association And Function Between Genetic Variants Of MiRNA-related Genes And Bladder Cancer Risk And Recurrence In The Chinese Populations

Bladder cancer is the most common urinary malignancy. Bladder cancer is the seventh most common cancer in men and the17th most common cancer in women worldwide. In China, the incidence and mortality of bladder cancer ranks the first of urinary malignancy (the ratio of male to female is4:1). The pathological type of bladder cancer generally includes transitional cell carcinoma, squamous cell carcinoma and adenocarcinoma, of which transitional cell carcinoma is more than90%. At present, cytoscopy is the main method to dignosis of bladder cancer. The difference between bladder cancer and other tumors is easy to recurrent. The development of bladder cancer is a complicated, multifactorial process, due to the unclear pathogenesis of bladder cancer. At present, the frequnce of bladder cancer recurrence is very high, making it hard to prevent and intervent the bladder cancer risk. Smoking and occulational exposure have been confirmed as the major risk factor of bladder cancer. Additionally, many studies have suggested that parents suffering from bladder cancer, the bladder cancer risk of their children will be greatly increased. Many studies also propose that individual susceptibility plays an important role in tumor development. Although many people have been exposed to the same risk factors, only a fraction of exposed individuals will develop the bladder cancer in their life, suggesting that there is the inter-individual variation in genetic susceptibility to bladder cancer.Human genome project (HGP) has confirmed that genetic variations play the crucial role in gene structure and function, which can alter the biological function of genes. SNPs (Single nucleotide polymorphisms) are the most common types of genetic variations. SNPs can influence the structure and expression of genes, thus affecting the regulation. Our previous studies have found that p53-mediated DNA damage stress response (MDM2, P53), DNA repair (XRCC1, APE1, ADPRT), apoptosis (CASP8, FAS, FAL), immune and inflammatory (IL-4, IL-4R, IL-13) and folate metabolism (MTHFR, MTR, MTRR), and other biological pathway gene SNPs were significantly associated with bladder cancer risk. Recently, genome wide association study has been used to investigate the association between SNPs and bladder cancer risk. In European populations, Kiemeney and Wu et al. have identified many bladder cancer-related susceptibility loci, for example rs9642880in8q24, rs710521in3q28, rs2294008in PSCA. So far, it has identified thirteen European bladder cancer related SNPs. It is suggested that individual genetic susceptibility indeed play an important role in the development of bladder cancer.MiRNAs (microRNAs) are a class of small noncoding RNAs (18-23nt), which play the roles in numerous processes, including development, proliferation, apoptosis and carcinogenesis. Studies have found that SNP singly or jointly can contribute to carcinogenesis, and possibly have gene-gene and gene-environment interactions. In2008, Hu and his colleagues had observed that miR-196a2rs11614914can influence the process of pre-miRNA to the mature miRNA, and this SNP was associated with lung cancer risk. However, there are few studies to explore the association between miRNAs SNPs and bladder cancer risk. Recently, many studies have shown that the abnormal expression of miRNAs are related with the development of bladder cancer: if SNP occured in the sequence of pri-miRNA (primary miRNA), it may affect the formation of the pre-miRNA (precursor miRNA) and mature miRNA; SNP is present in the miRNA biological processing key gene (DICER and DROSHA), it may influence the expression and function of miRNA biological processing gene, resulting in alted expression of mature miRNA. As we all known, miRNAs can regulate gene expression at the post-transcriptional level through binding the3’UTR of target gene mRNA. Thus, if SNP located in the sequence of mature miRNA, it may affect the binding activity of miRNA and target gene; if SNP located in or near the binding site of target gene and miRNA, likewise, SNPs may affect the binding activity of miRNA and target gene. Currently, miRNAs have been extensively studied, however, the biologic function of miRNAs in bladder carcinogenesis are not fully clear. In the present study, we focued on investigating the association and function between genetic variants of miRNAs, miRNAs target gene and miRNAs biological processing gene and bladder cancer risk and recurrence in the Chinese population.Part I Genetic variants in miRNAs predict bladder cancer risk and recurrenceRecently, MiRNAs play important roles in carcinogenesis. In this study, we investigated whether genetic variations in miRNAs are associated with bladder cancer risk and prognosis. First, we queried all known human miRNA available in the public miRBase database and collected564miRNAs, then, we conducted extensive searches of SNP databases including HapMap, dbSNP, and Patrocles, and the Gibbs binding free energy (DG, kJ/mol) for each pair of common and variant alleles was computed using miRanda software. Finnally, we selected five SNPs located in miRNAs (miR-146a rs2910164、miR-196a2rs11614913、miR-605rs2043556、miR-618rs2682818和miR-923rs47960429).We used these SNPs to evaluate miRNA-disease associations in a two-stage model, consisting of1,019bladder cancer cases and1,182controls (683cases and728controls in the training set and336cases and454controls in the test set). In the first stage, we observed that rs2910164in miR-146a and rs2682818in miR-618were significant (adjusted OR=0.80,95%CI=0.69-0.93, P=0.004for miR-146a rs2910164and adjusted OR=0.81,95%CI=0.69-0.96, P=0.013for miR-618rs2682818in the additive model). But this significance disappeared after Bonferroni correction for miR-618rs2682818(P=0.064). Furthermore, in the test set, only miR-146a rs2910164showed a significant association with bladder cancer risk (P=0.030), consistent with the results fromthe training set analysis. In the combined set analyses, the rs2910164C allele in miR-146a was associated with20%decreased risk of bladder cancer, compared with the G allele (adjusted OR=0.80,95%CI=0.71-0.90, P=2.920×10-4). When we conducted stratified analyses by age, sex, smoking status, grade, and stage, we observed a decreased bladder cancer risk for the rs2910164C allele; this was more pronounced among male subjects (adjusted OR= 0.76,95%CI=0.66-0.87). In addition, compared with G allele, the individuals with C allele can reduce the risk of bladder cancer among old (adjusted OR=0.78.95%CI=0.66-0.92) or young (adjusted OR=0.82,95%CI=0.69-0.97), smoker (adjusted OR=0.80,95%CI=0.67-0.96) or non-smoker (adjusted OR=0.80,95%CI=0.69-0.93), tumor grade (G1/G2:adjusted OR=0.83,95%CI=0.73-0.94; G3:adjusted OR=0.66,95%CI=0.52-0.85), tumor stage (superficial bladder cancer:adjusted OR=0.78,95%CI=0.68-0.89; invasive bladder cancer:adjusted OR=0.83,95%CI=0.70-0.99). We further investigated whether there was any association between miR-146a rs2910164genotypes and the recurrence of nonmuscle invasive bladder cancer in the test set that had clinical follow-up data (119). Compared with the rs2910164GG genotype, a significant overall42%decrease in recurrence risk was observed for individuals carrying at least1rs2910164C allele (HR=0.58,95%CI=0.36-0.94, P=0.016). In an attempt to determine the functional role of miR-146a rs2910164in bladder cancer carcinogenesis, we found that miR-146a s2910164G>C can affect bladder cancer cell proliferation by target with IRAKI and TRAF6. We also evaluated the association between miR-146a rs2910164and miRNA expression levels in64bladder cancer tissues detected by real-time quantitative RT-PCR, and results showed that the rs2910164C allele was significantly associated with increased expression of miR-146a (P=0.010). Taken together, our findings indicate that miR-146a rs2910164plays important roles in the risk and recurrence of bladder cancer. Futher larger and prospective cohorts are needed to validate our findings.Part Ⅱ Genetic variants in miRNAs target gene EGFR3’UTR predict bladder cancer riskGenetic variants in3’UTR of gene have been reported to affect gene expression by interfering with miRNAs. Many evidences confirm that over-expression of epidermal growth factor receptor (EGFR) plays an important role in regulating carcinogenesis. Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor widely expressed on the surface of epithelial cells, and it modulates some important processes in carcinogenesis by mediating cell mortality, apoptosis, tumour invasion and metastasis. One polymorphism in EGFR3’UTR, a T to C transition at774-bp (774T>C, rs884225), which is located in the binding region of miR-214. Thus, we considered that this SNP may influence the binding activity of miR-214and EGFR mRNA3’UTR, and then affected post-transcriptional of EGFR. In this study, we investigated the association between the EGFR3’UTR774T>C polymorphism and bladder cancer risk. We used the TaqMan method to genotype this genetic variant in a hospital-based case-control study of908bladder cancer patients and1,239controls in a Chinese population. We found that the774CC genotype was associated with a statistically significantly increased risk of bladder cancer (adjusted OR=1.29,95%CI=1.05-1.58), comparing with the774TT/TC genotype, and this increased risk was more pronounced among subgroups of age>65years (adjusted OR=1.87,95%CI=1.39-2.53), non-smokers (adjusted OR=1.46,95%CI=1.11-1.92), tumor grade2(adjusted OR=1.34,95%CI=1.01-1.80), and tumor invasive stage patients (adjusted OR=1.40,95%CI=1.05-1.89). To test whether miR-214targets EGFR3’UTR, we first generated reporter constructs through replacing the3’UTR of a Renilla luciferase reporter gene with the full-length3’UTR of EGFR containing either774T or774C allele of EGFR3’UTR. We generated a reporter construct that included18-bp deletion of putative miRNA target (774D). Then, we used them for transient transfections with the T24cell (NC, negative control miRNA or miR-214). For NC miRNA transfection, the construct with the774T allele had a relatively decreased luciferase activity, compared with others with the774C allele and18-bp deletion in T24cell (P<0.05). Likewise, we observed that the luciferase activity from construct including the774T allele was significantly reduced in the presence of miR-214(C vs. T,P<0.05; D vs. T,P<0.05). Comparing with NC, we observed that miR-214did not affect luciferase activity. Taken together, our data suggest that EGFR3’UTR774T>C may influence EGFR expression, which was not modulated by miR-214. However, the exact mechanism needs to be further clarified. These findings suggest that EGFR3’UTR774T>C polymorphism may contribute to the susceptibility of bladder cancer, especially pronounced among old, non-smokers, tumor grade2and tumor invasive stage patients.Part III Genetic variants in miRNAs biological processing key genes predict bladder cancer riskDICER and DROSHA are two members of RNase III family, playing pivotal roles in the pathway of miRNAs biogenesis. In this study, we hypothesized that genetic variations of the DICER and DROSHA genes were associated with the bladder cancer risk. We performed a case-control study of685bladder cancer cases and730controls to investigate the association between the seven functional SNPs of DICER (rsl2323635C<T, rs13078T>A, rs1057035T>C, and rs3742330A>G) and DROSHA genes (rs2291109A>T, rs10719T>C, and rs642321C>T) and bladder cancer risk. We then evaluated the functionality of the important SNPs. Results suggested that there was no significant association between DICER rs12323635C>T, rs13078T>A, rs1057035T>C, rs3742330A>G, DROSHA rs2291109A>T, rs642321C>T and bladder cancer risk (all P>0.05). We only observed that rs10719T>C polymorphism located in3’UTR of DROSHA gene was associated with the increased risk of bladder cancer (adjusted OR=1.25,95%CI=1.01-1.55, P=0.041, TC/CC vs. TT). Furthermore, stratified analysis suggested that rs10719TC/CC genotype can increase risk of bladder cancer among male patients (adjusted OR=1.34,95%CI=1.05-1.70, P=0.018), and ever smokers (adjusted OR=1.56,95%CI=1.14-2.14,0.006), compared with TT genotype. Based on bioinformatics analysis, we predicted that miR-27a/b can bind with the3’UTR region of DROSHA by using four common websites (TargetScan, miRanda, Microcosm, and PITA). Here, we chose J82and T24cell lines in the further luciferase assay. Then, we used the plasmids for transient co-transfection with the T24cells (stable NC miRNA:NC or miR-27a/b mimics) and J82cells (inhibitor NC or miR-27a/b inhibitor). In T24cell, miR-27a/b suppressed luciferase expression in the presence of rs10719T allele, comparing with NC (P<0.05), but not the rs10719C allele (P>0.05). Additionally, we also found that the inhibition of miR-27a/b expression can increase luciferase expression efficiently for the rs10719T-containing plasmid rather than the rs10719C-containing plasmid in J82 cells (P<0.05). However, we also found that a significant decrease in luciferase expression, when we added miR-27a inhibitor to allele C in J82cells. The similar result did not be observed in adding miR-27b inhibitor to allele C in J82cells. Maybe, miR-27a did not directly affect DROSHA luciferase expression. Our data suggested that rs10719T to C substitution acts as a loss-of-function mutation and would affect DROSHA luciferase expression by miR-27b target. Taken together, these findings suggest that DROSHA rs10719T>C polymorphism may be associated with bladder cancer risk in a Chinese population, and miR-27b can influence the expression of DROSHA protein by binding with3’UTR.