| Part I Analysis of LSP1 single nucleotide polymorphisms(SNPs) on bladdercancer risk in a Chinese population Objectives: To validate the association between LSP1 single nucleotide polymorphisms and bladder cancer risk in a Chinese population. Materials and Methods: A total of 535 bladder cancer patients and 649 health controls were recruited for our study from the Second Hospital of Tianjin Medical University, Tianjin, North China. Peripheral venous blood sample was obtained for each subject after the interview. Genotyping of the SNPs performed using ligation detection reaction-PCR(LDR-PCR). Hardy–Weinberg equilibrium was evaluated by a goodness-of-χ2 test to compare the observed genotype frequencies with the expected ones of the controls. Unconditional multivariate logistic regression was applied to calculate odds ratios(ORs) and 95% confidence intervals(CIs) to estimate the association between the polymorphisms and susceptibility of bladder cancer, with adjustment for age, sex and smoking status. Kaplan-Meier estimates and log-rank test were obtained to analyze the association between the genotype and risk of recrudesce in nonmuscle-invasive bladder cancer patients. Results The genotype distributions for rs907611 in controls were in accordance with the predicted from the Hardy–Weinberg equilibrium model(P>0.05). The A/A genotype had a significant association with increased bladder cancer risk(p=0.036,OR=1.786,95%CI 1.038-3.072, compared with G/G genotype. The combined group(G/A+A/A genotype) had a moderate association with increased bladder cancer risk as well(p=0.049,OR=1.264,95%CI 1.001-1.596); Compared to the G allele of rs907611, the A allele implicated a significantly increased risk for bladder cancer susceptibility(p=0.019,OR=1.264,95%CI 1.038-1.538). Stratified analysis results showed that showed that there was no assocaiation between rs907611 and grade/stage of bladder cancer. The rs907611 polymorphism has no effect on the incidence rate of bladder cancer in smoking population. No assocaiation was found between the genotype and risk of recurrence in nonmuscle-invasive bladder cancer patients. Conclusions The rs907611 polymorphism is associated with risk of bladder cancer in a northern Chinese population, especially for the low grade and low stage of bladder cancer. However,it does not affect the incidence rate of bladder cancer in smoking population. No assocaiation was found between the genotype and risk of recurrence in nonmuscle-invasive bladder cancer patients.Part II Function analysis of rs907611 Objective: we aimed to explore the mechanism of rs907611 polymorphism predisposes to bladder cancer by assess the impact of risk allele on expression of the gene in close vicinity of rs907611. Methods: We performed an in silico search for genes adjacent to rs907611 in the UCSC Genome browser. Genotype of rs907611 was investigated in 24 pairs of bladder cancer tissues and paired adjacent non-cancerous tissues. The expression level of genes near to rs907611 was analysis by q RT-PCR. The association of these genes expression level and rs907611 genotype was evaluated by Chi-square test. Results: There were 21 genes closet to rs907611 in all( KRTAP5-3、KRTAP5-4、KRTAP5-5、KRTAP5-6、IFITM10、SYT8、TNNI2、LSP1、CTSD、TNNT3、HOTS and MRPL23;MIR4298、MIR7847 and MIR675; MRPL23-AS1、FAM99A、FAM99B、H19、Linc01219 and Linc01150). However, no association were found between rs907611 genotype and the expression levels of these genes(P>0.05). Interestingly, LSP1 and MIR7847 were down-regulated in bladder cancer tissues(P<0.05), while the expression level of H19 and MIR675 was significantly higher in bladder cancer tissues compared to that in adjacent noncancerous tissues(P<0.05). Conclusion: The genotype of LSP1 polymorphism had no effects on the expression of its neighboring genes.LSP1 and MIR7847 levels in cancerous tissues were evidently lower than those in the noncancerous tissues, while H19 and MIR675 were up-regulated in bladder cancer tissues. Part III Clinical Significance of LSP1 Expression in Human Bladder cancer and function anlysis of LSP1 Objective: To investigate the expression of LSP1 in bladder cancer and evaluate the correlation between LSP1 expression level and clinicopathological variables; illuminate the functions of LSP1 in bladder cancer. Methods The expression level of LSP1 between bladder cancer tissues and paired adjacent non-cancerous tissues were detected by IHC and western blot. The function of LSP1 was assessed by overexpression LSP1 in bladder cancer cell lines. Total RNAs and proteins were colleted after transfection, further q RT-PCR and western blot were used to analysis the expression level of LSP1 m RNA and protein. The function of bladder cancer cell proliferation was assessed by MTT.The apoptotic rate was detected by flow cytometry. The migration and invasion of bladder cancer cells were investigated by wound healing assay and transwell assay. Results: The expression level of LSP1 was down-regulated in bladder cancer tissues. MTT assay showed that the proliferation of T24 cells remarkably inhibited after LSP1 overexpression. The results of Flow cytometry shows that overexpression of LSP1 promotes apoptosis of T24 cells. Wound healing assay and transwell assay showed that high expression of LSP1 inhibit migration and invasion of T24 cells. Conclusions: LSP1 was down-regulated in bladder cancer tissues. Over expression of LSP1 in bladder cancer cells inhibits cell proliferation, invasiveness and promote apoptosis in vitro. |
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