The Preliminary Association And Molecular Mechanism Study Of LncRNA SNHG16 Genetic Variations And Bladder Cancer Risk

Background:Bladder cancer（BCa）is one of the most frequently occurring urological malignancies in the world.In China,the incidence of BCa ranks first among male urinary tract cancers,and it is still rising.Due to the lack of early clinical features of BCa.Most patients have been suffering in the advanced stage.Although the current level of medical treatment and surgical chemotherapy have increased significantly,there is still a relatively high recurrence rate and metastasis rate in BCa,which impacts the quality of patients’life and brings serious social burdens.Therefore,it is very important to strengthen the molecular mechanism study of BCa and screen highly effective and sensitive biomarkers to identify early BCa.Single nucleotide polymorphism（SNP）is the most common genetic variation,which has a significant effect on tumor susceptibility.Long non-coding RNAs（lncRNAs）also play an important role in the development and progression of tumors.Therefore,more and more tumor projects have used lncRNAs as candidate genes for SNPs research and have achieved satisfied results.However,there is still a lack of research on molecular mechanism of lncRNAs genetic variations and BCa risk.Former study found that SNHG16 expression levels in BCa tissues and cell lines were higher than those in adjacent tissues and normal cells.SNHG16 overexpression in BCa cells significantly increased its proliferation,invasion,and metastasis ability,suggesting that SNHG16 played an oncogene role in the development and progression of BCa.However,there is still no research about lncRNAs genetic variations on BCa risk and molecular mechanism.This study would choose SNHG16,an oncogene in BCa,as a candidate lncRNA for the following study.Methods:We used 1000 Genomes Project to search for all SNPs between 2000bp upstream and downstream of lncRNA SNHG16,and a standard quality control procedure was performed on these SNPs.We genotyped these SNPs in 578 BCa cases and 1,006 normal controls using Illumina Human Omni chip,and then we validated the association between candidate SNPs and BCa risk by TaqMan genotyping experiment in 1,050 cases and 1,404 normal controls.We used expression Quantitative Trait Loci（eQTL）to analyze the expression level of SNHG16 under SNP different genotypes.To explore its specific molecular mechanism as a ceRNA,we detected the Nuclear-cytoplasm localization of SNHG16 in BCa cells,and then studied the effect of different SNHG16 expression levels on BCa by molecular biology methods.Results:We screened three SNPs rs4647885,rs12952443,rs12944474 with statistically significant differences（P<0.05）in BCa and normal controls by Illumina Human Omni chips,and there was a high linkage disequilibrium（LD）between these three SNPs.In this study,we chose rs4667885,which had the smallest P value(P=7.81×10^-3)and genotyping for TaqMan genotyping experiment.After adjusting for age and sex,rs4647885 was significantly associated with BCa risk in the dominant model,and significantly reduced BCa risk when carrying the C allele[odd ratios,（OR）=0.82,95%confidence interval（CI）=0.68-0.99,P=0.046].In the codominant heterozygote model,it was also found that carrying TC genotype significantly reduced BCa risk compared with carrying TT genotype（OR=0.79,95%CI=0.65-0.97,P=0.022）.By stratified analysis,we found that rs4647885 carrying C allele significantly reduced BCa risk（OR=0.73,95%CI=0.58-0.93,P=0.009）in the group older than 60 years after adjusting for age and sex.When carrying C allele（TC+CC）,the expression of SNHG16 was significantly lower than only carrying T allele（TT）by eQTL.SNHG16 was located in the cytoplasm significantly higher than in the nucleus.It could play a ceRNA role to competitively bind miR-140-5p,thereby increasing the expression level of GIT1 to affect the development of BCa.Conclusion:SNHG16 rs4647885 carrying C allele significantly reduced BCa risk.The specific molecular mechanism might be that rs4647885 C allele reduced SNHG16 expression,thereby inhibiting its competitive binding to miR-140-5p and decreasing GIT1 expression.