去泛素化酶COPS5稳定m6A结合蛋白YTHDC1促进肿瘤EMT及转移的机制研究

转移是晚期结直肠癌致死的主要原因。我们前期研究发现m6A结合蛋白及剪接复合体组分YTHDC1在转移性结直肠癌中高表达，转录组和m6A测序结果表明YTHDC1可调控转移相关基因ZO-1表达。课题组通过对82种去泛素化酶的siRNA筛选，发现基因沉默COPS5对YTHDC1蛋白下调最明显，蛋白质谱分析提示COPS5与YTHDC1相互作用。而且，COPS5和YTHDC1的蛋白表达与结直肠癌细胞的侵袭转移能力呈正相关。由此推测，结直肠癌中COPS5通过去泛素化稳定YTHDC1，促进其对靶pre-mRNA异常剪接而产生“癌性”异构体，促进癌细胞EMT和转移。本项目将利用泛素化、RIP、剪接报告基因等分子生物学及动物实验，并结合临床组织标本，阐明COPS5稳定YTHDC1影响结直肠癌细胞EMT和转移的机制。本项目的实施将为开发靶向YTHDC1的临床抗肿瘤治疗策略提供理论依据和新思路。

Metastasis is the leading cause of cancer-related death in advanced colorectal cancer (CRC). Our pilot data revealed that the m6A binding protein YTHDC1, which is a component of the spliceosome, is highly expressed in metastatic colorectal cancer cells. The transcriptome-sequencing and m6A-sequencing analysis showed that the tight junction protein ZO-1 is a candidate target gene which is regulated by YTHDC1. By using an 82 deubiquitinases-targeting siRNA library screening, we found that knockdown of deubiquitinase COPS5 results in the most significant reduction of the nuclear protein YTHDC1. Furthermore, the MS/MS data indicated that COPS5 interacts with YTHDC1. In addition, both COPS5 and YTHDC1 are highly expressed in CRC cell lines and positively correlated with the metastatic capacity of CRC cells. These results suggested that COPS5 might regulate YTHDC1 stability through deubiquitination and further modulate YTHDC1-mediated alternative splicing of the pre-mRNA of its target gene, which inducing “cancerous” splicing variant formation, and ultimately driving epithelial-mesenchymal transition (EMT) and metastasis. The in vitro and in vivo assays (including RNA-binding protein immunoprecipitation, splicing reporter minigene assay, and in vivo xenograft mouse model) will be performed to elucidate the mechanisms of how COPS5-stabilized YTHDC1 regulates alternative splicing to promote EMT and metastasis in CRC cells. Thus, targeting YTHDC1 turnover might be a promising approach for metastatic colorectal cancer treatment.