circRNA_40753以 “protein sponge”方式结合APC/β-catenin蛋白复合物促进胃癌发生发展的作用机制

Recently, circRNA has been shown to act as a sponge for miRNAs and play an important role in oncogenesis, however the other functional regulatory mechanism of circRNA is not clear. Based on the preliminary data, circRNA_40753 was screened as an oncogenic circRNA in gastric cancer. CircRNA_40753 promotes gastric cancer cell growth and migration. Based on the bioinformatic analysis and preliminary results, we propose a new regulatory mechanism of circRNA_40753 acting as “protein sponge”: namely circRNA_40753 contributes to the carcinogenesis and development of gastric cancer by activating Wnt signaling pathway via sponging APC from interacting with APC/β-catenin complex. .Firstly, we will evaluate the biological impact of circRNA_40753 in vitro and in vivo by transfection the overexpression plasmid or siRNA into cells followed by migration, invasion, proliferation, cell cycle, apoptosis assays, EMT and animal study. Secondly, we will confirm the interaction of circRNA_40753 with APC/β-catenin complex using FISH、RPI、RNA pull down as well as confocal immunostaining. Finally, we will explore the mechanism by which circRNA_40753 activates Wnt pathway using luciferase assay，western blot, real time PCR, CHIP, COIP and so on. The study will ultimately reveal a new regulatory mechanism of circRNA_40753 and clarify the mechanism by which circRNA_40753 promotes gastric carcinogenesis. It will ultimately provide novel circRNA molecular markers and targets for clinical diagnosis and tailored treatment of gastric cancer.

近期研究表明环状RNA可作为“miR海绵”介入肿瘤演进，但其是否存在其他作用机制亟待研究。预实验筛选出circRNA_40753为一个致癌环状RNA，能促进胃癌细胞的生长和迁移；根据生物信息学分析及预实验结果，我们提出了一个新的功能机制：即circRNA_407533通过与APC/β-catenin蛋白复合物的直接结合，“protein sponge”APC，促进β-catenin入核进而激活Wnt信号通路。本项目拟利用多种细胞分子生物学方法通过体内体外实验阐明这一机制。最终探明（1）circRNA_40753在胃细胞中的生物学功能；（2）其是否结合APC/β-catenin蛋白复合物；（3）circRNA_40753是否激活Wnt通路。旨在揭示一个新的环状RNA“protein sponge”功能机制，为临床胃癌诊治提供新的环状RNA分子标志物和靶点。

环状RNA (circRNA)是一类具有共价闭合环状结构的内源性RNA。最近研究表明，circRNA参与肿瘤发生和发展。本课题旨在鉴定在胃癌中异常表达的环状RNA，并研究circRNA在胃癌发生发展过程中发挥的重要作用以及作用机制。我们发现来源于人癌胚抗原相关细胞粘附分子5基因的circRNA，circRNA_40753在胃癌中发挥致癌驱动因子的作用。 circRNA_40753在胃癌组织及细胞系中显著高表达。在胃癌细胞系中下调circRNA_40753之后，细胞增殖、迁移和克隆形成受到抑制，表明在胃癌中该circRNA可能具有促癌的作用。此外，下调circRNA_40753能促进血清饥饿和Doxorubicin（Dox）等刺激诱导的细胞凋亡。我们利用RNA pull down 和蛋白质谱技术鉴定circRNA_40753可能与之相互作用的蛋白，并发现circRNA_40753可能与调控凋亡的蛋白p53以及与p53磷酸化相关的激酶CDK1结合。我们进一步利用RNA Binding Protein Immunoprecipitation 和 RNA pull down技术证实了该环状RNA与p53和CDK1的结合。据报道，CDK1介导p53在s315位点的磷酸化。我们发现circRNA_40753的下调减少了Dox诱导的p53在s315位点的磷酸化，并且增加了细胞核内p53的水平和活性，进一步增加p53靶基因的表达。由此，本研究揭示了circRNA_40753参与胃癌发生发展的新机制，即circRNA_40753通过与p53和CDK1蛋白相互作用，抑制细胞凋亡，从而保护胃癌细胞免于血清饥饿和Dox等刺激诱导的细胞凋亡。该项目的研究不仅有助于阐明胃癌发生发展的分子机制，并且为寻找新的胃癌治疗靶点提供科学依据。