RAC1基因3’端非编码区的选择性多聚腺苷酸化在膀胱癌中的作用与临床意义

Cancer cell lines and tissues often expressed substantial amounts of mRNA isoforms with shorter 3’ untranslated regions (UTRs), resulting from alternative cleavage and polyadenylation (APA) regulation. The APA had functional consequences, with the shorter mRNA isoforms of certain oncogene leading to far more oncogenic transformation than did expression of the full-length, annotated mRNA. Moreover, selected APA events add strong prognostic power beyond common clinical and molecular variables, suggesting their potential as novel prognostic biomarkers. Bladder cancer (BC) is one of the commonly diagnosed cancers in the world and has the highest rate of recurrence of any malignancy. Thus, identification of novel biomarkers for prognosis will help to advancer clinical diagnosis. With our preliminary data, it showed that RAC1, a proto-oncogene, expressed shorter 3’UTR isoform in BC of our sequenced Chinese patients, as well as in USA patients from TCGA database. Statistic analysis from BC clinical samples shows that shorter 3’UTR of RAC1 is associated with poor clinical outcome and RAC1 protein expression. Further functional study showed that overexpressing shorter 3’UTR of RAC1 in T24 cell line, led to produce ten folds of protein and enhanced cell proliferation. We assumed that the difference in protein expression might result from loss of miR-182 repression in shorter 3’UTR of RAC1 . To further investigate the biological function of shorter 3’UTR of RAC1, and its regulatory mechanism in BC, we plan: 1) Reveal the clinical significance of shorter 3’UTR of RAC1 in more BC samples; 2) Using multiple cell lines and animal models to study its biological function; 3) Investigating the regulatory role of miR-182 target site from full length 3’UTR of RAC1 in BC. Therefore, our study may suggest that RAC1 gene activation and overexpression is due to escape of the shorter isoform from miR-182-mediated repression. And it may reveal that shorter 3’UTR isoform of RAC1 might serve as a novel prognostic biomarker in BC.

肿瘤中很多原癌基因通过选择性多聚腺苷酸化（APA）的调控，表达3’UTR缩短的mRNA，具有显著增加的克隆形成能力，与不良预后密切相关，而APA调控在膀胱癌中的作用尚未报道。膀胱癌是泌尿系统最常见的恶性肿瘤，其发生、发展机制尚未清楚。申请者前期通过分析中国膀胱癌患者和美国TCGA的RNA测序数据，发现RAC1在膀胱癌中多表达3’UTR缩短的转录本，与临床预后呈负相关。进一步研究表明：在膀胱癌T24细胞系中，RAC1短3’UTR转录本可促进蛋白表达和体外增殖，其作用机制可能是由于缺失了miRNA的靶向位点，逃逸了miR-182的抑制作用。本研究拟在更多的膀胱癌组织样本中，明确RAC1短3’UTR转录本的临床意义和价值，利用多个膀胱癌细胞系和动物模型，验证其生物学作用，并阐明其机制是通过逃逸了miR-182的靶向抑制，从而影响RAC1的蛋白表达。本项目的完成，可能为膀胱癌提供新的临床预后指标。

肿瘤中很多原癌基因通过选择性多聚腺苷酸化（APA）的调控，表达3’UTR缩短的mRNA，具有显著增加的克隆形成能力，与不良预后密切相关，而APA调控在膀胱癌中的作用尚未报道。膀胱癌是泌尿系统最常见的恶性肿瘤，其发生、发展机制尚未清楚。申请者通过分析中国膀胱癌患者和美国TCGA的RNA测序数据，发现RAC1在膀胱癌中多表达3’UTR缩短的转录本，与临床预后呈负相关。进一步研究表明：在膀胱癌T24细胞系中，RAC1短3’UTR转录本可促进蛋白表达和体外增殖，其作用机制可能是由于缺失了miRNA的靶向位点，逃逸了如miR-182的抑制作用。本研究发现RNA加工因子CSTF2能直接调控RAC1 3’UTR的缩短。CSTF2通过结合在近端的polyA位点GUAAU motif，抑制了转录延伸因子AFF4和AFF1的结合，抑制了转录延伸，生成短RAC1转录本。CSTF2通过调控RAC1 3’UTR的缩短，促进膀胱癌的增殖和侵袭转移。在膀胱癌病人的肿瘤标本中，CSTF2的高表达与RAC1 3’UTR 缩短的转录本，与临床病人预后差成正比。本项目的完成，可能为膀胱癌提供新的临床预后指标。