SRSF10-SREK1可变剪接信号轴在肝癌形成和发展中的功能及分子机制

Hepatocellular carcinoma (HCC) is one of the most common cancers in China. The mechanism of the initiation of HCC is complicated. Aberrant mRNA alternative splicing can cause the inactivation or over-activation of vital regulatory proteins in liver cells, directly leading to the malignancy of liver cells. However, currently little was known for the HCC-associated alternative splicing events and regulatory mechanisms. Based on our previous transcriptome analysis of HCC patients’ tissues, the applicant found that the exon 10 of SREK1 gene is alternatively spliced between tumor tissue and match normal tissues. The expression of longer variant of SREK1 gene (SREK1L, containing exon10) is significantly upregulated in HCC tumor tissues than in match normal tissues. This project aims to investigate the biological function of SREK1L and its potential splicing regulator - SRSF10 in HCC cells and mice models; to explore the molecular regulatory mechanisms of SREK1L in HCC, to identify the proteins interacting with SREK1L or SRSF10, to analyze the signalling network of the interactome, to eventually reveal the function and molecular mechanism of SRSF10-SREK1L signalling axis in the initiation and development of HCC and to explore the axis-related small molecular inhibitors via molecular cellular biological, proteomic and transcriptomic analysis. This study helps to understand theoretically how the aberrant alternative splicing network contributing to the initiation and development of HCC and further offers a novel therapeutic target for the development of small inhibitors in future.

肝癌是我国高发的癌症之一，其发病机理复杂，肝细胞内mRNA异常可变剪接会造成关键调节蛋白的失活或超活化，直接导致肝细胞癌化。然而，目前肝癌相关的可变剪接事件和调控机制研究还不足。通过对肝癌病人组织的转录组测序，申报人发现肝癌中SREK1的10号外显子发生了异常的可变剪接，SREK1全长变体（SREK1L）的基因表达在肝癌中较癌旁组织显著地上调。本项目拟通过肝癌细胞和动物模型来评价SREK1L及其潜在的剪接调节因子SRSF10的生物学功能；利用分子细胞生物学、转录组学和蛋白质组学等技术揭示SREK1L的分子调控机理，筛选SREK1L和SRSF10的相互作用蛋白，分析其参与的信号网络，最终阐述SRSF10-SREK1L信号轴在肝癌形成和发展中的功能和分子机制，并探索相关的小分子抑制剂。这将在理论上帮助理解异常的可变剪接网络如何促进肝癌的形成和发展，为今后研发肝癌的小分子抑制剂提供新的靶点。

背景与研究目标：可变剪接异常调控是癌症异质性的重要原因之一，但在肝细胞癌化过程中关键的可变剪接靶点与内在驱动机制或网络还未知。本研究拟揭示SREK1变体及其上游剪接调控因子SRSF10上述过程中扮演的重要功能。.方法：通过聚合酶链式反应（PCR）检测对肝癌组织中包含和剔除10号外显子的SREK1变体（分别简称为：SREK1L和SREK1S）进行表达验证并与患者临床预后进行分析。通过肝癌细胞系和小鼠模型对SREK1L变体的体内、外功能以及调控机制进行深入研究。通过PCR、免疫印迹和免疫荧光等方法，对SRKE1L上、下游信号及靶点进行深入研究。.结果：高的SREK1L与SREK1S变体表达比例与预后较差的肝癌显著相关。细胞动物实验证实SREK1L在肝癌中发挥原癌基因的作用，促进肝癌细胞的增殖、“干样”、迁移以及成癌等特性。机理研究实验发现SREK1L可以通过抑制无义介导的RNA降解（NMD）信号EJC复合物对NMD靶点BLOC1S5-TXNDC5 (B-T)的识别与降解，进而促进非编码RNA B-T的表达。深入研究发现作为SREK1L下游关键效应因子，B-T发挥其内源竞争RNA（ceRNA）的功能，抑制miR-30c-5p和miR-30e-5p的表达，促进下游原癌基因SRSF10和TXNDC5的表达。有趣的是，随后实验揭示SRSF10参与形成一个剪接调控复合物，并作为SREK1L的上游剪接调控因子，促进SREK1L的表达并发挥促癌效应。.结论：本研究揭示了一个新的 SRSF10/SREK1L/B-T正循环信号，通过调控可变剪接与ceRNA信号网络，加速肝癌形成。实验还表明SRSF10是一个潜在的、有价值的抗肿瘤药物靶点。