lncRNA选择性剪接(AS)遗传变异与膀胱癌易感性及其机制研究

Bladder cancer (BCa) is a common malignant cancer in the urinary system with a high recurrence rate, however, the mechanism of BCa remain largely obscure. Emerging evidence has shown that aberrant alternative splicing (AS) is involved in the carcinogenesis of cancers. Furthermore, single nucleotide polymorphisms at alternative splicing site (AS-SNPs) were reported to be associated with susceptibility of cancer through regulating the expression of splicing isoforms and contributing to aberrant splicing event. Through integrative analysis of database of Single Nucleotide Polymorphisms (dbSNP) and genome-wide association study (GWAS), we identified that seven candidate AS-SNPs located at long non-coding RNA (lncRNA) were associated with BCa risk. Taken together, AS-SNPs of lncRNA are hypothesized to affect the process of AS and result in deregulation of target genes, thus involved in the development of BCa. On the basis of previous study results, we intend to further validate the associations between candidate lncRNA AS-SNPs and susceptibility of BCa in other independent case-control study. Besides, we apply molecular biology experiments to assess the effect of AS-SNPs on expression level of lncRNA isofoms and malignant phenotype of BCa. Furthermore, RNA pull-down and RNA immunoprecipitation (RIP) are used to explore the underlying mechanism by lncRNA isoforms confers regulation of target genes. Our study may provide beneficial information for etiology and screening of high-risk population of BCa.

膀胱癌是常见的泌尿系统恶性肿瘤，易复发，但机制不清。选择性剪接（AS）改变与肿瘤发生密切相关，其中剪接位点单核苷酸多态性（AS-SNP）可产生异常剪接异构体，是导致AS异常事件的重要因素。本项目前期通过dbSNP数据库结合全基因组关联性研究发现7个lncRNA AS-SNPs与膀胱癌发病风险显著相关。据此提出科研假说：lncRNA AS-SNPs与膀胱癌易感相关，可改变lncRNA AS过程，影响其对下游靶基因的调控，进而参与膀胱癌发生发展。本项目拟进一步通过病例-对照研究，分析候选lncRNA AS-SNPs与膀胱癌发病风险及复发的关联性；采用分子生物学方法评价AS-SNPs对lncRNA剪接异构体表达水平和膀胱癌恶性表型的影响；通过RNA pull-down结合RIP实验探讨lncRNA剪接异构体对靶蛋白的调控机制，为膀胱癌病因学研究及高危人群筛选提供科学依据。

选择性剪接（Alternative Splicing，AS）是生物体内普遍存在的基因表达调控方式，可使前体mRNA被剪接成多种异构体，是丰富蛋白产物的重要机制。研究发现，选择性剪接模式常发生改变进而参与肿瘤的发生发展，但选择性剪接相关的单核苷酸多态性（SNPs）在肿瘤发生发展中的潜在机制尚未完全阐明。本研究通过dbSNP数据库结合本课题组膀胱癌全基因组关联性研究，筛选出与AS相关的SNPs，并探讨其与膀胱癌发生发展的关联性及作用机制。结果发现，位于lncRNA BCLET的SNP rs558814 A>G可以降低中国人群膀胱癌的发病风险；rs558814 G等位基因对BCLET的转录活性显著高于A等位基因，SNP rs558814 A>G促进了BCLET转录本BCLET-long和BCLET-short的表达水平；BCLET在膀胱癌组织和细胞中表达水平显著降低，且BCLET-long和BCLET-short转录本的过表达均可抑制膀胱癌的细胞增殖、平板克隆、迁移侵袭和肿瘤生长，进一步促进细胞凋亡，提示BCLET发挥抑癌基因样作用；还发现lncRNA BCLET可识别并调控MSANTD2的选择性剪接事件，优先促进MSANTD2-004转录本的剪接率及表达水平上调，从而参与膀胱癌的发生发展。另外，通过膀胱癌病例-对照研究也发现剪接因子RBFOX3 SNP rs978416 G>A可降低中国人群膀胱癌的发病风险，RBFOX3 mRNA在膀胱肿瘤组织中表达水平降低，且RBFOX3的高表达与较高的膀胱癌分期密切相关；SNP rs4383 C>G可降低中国人群膀胱癌发病风险，rs4383与其下游基因MAFF的可变5'剪接位点的选择性剪接事件相关，MAFF在膀胱癌组织中表达水平下降，且与不良预后密切相关。本研究系统探讨了选择性剪接相关遗传变异参与膀胱癌的关联性和可能的潜在机制，可为膀胱癌早期筛查、个体化预防以及阐明膀胱癌发病机制提供新的线索。