肝癌关键可变剪接因子LSM4的功能及其分子调控机制研究

肝癌是一种高致死率的恶性肿瘤，异常的RNA可变剪接和肝癌发生发展息息相关。然而，肝癌增殖相关可变剪接因子还有待系统筛选。我们利用CRISPR-Cas9技术对肝癌细胞增殖必需可变剪接因子进行筛选，发现U6剪接体组分LSM4是肝癌增殖最为关键的一个剪接因子。转录组分析表明其通过调控MYO1B24号外显子等靶点可变剪接来发挥促癌功能。本项目拟通过肝癌细胞、小鼠和肝癌组织等模型来评价LSM4及MYO1B剪接变体MYO1B-L和MYO1B-S的表达、定位和生物学功能；利用细胞分子生物学、转录组学和蛋白质组学等技术揭示LSM4和MYO1B-L促肝癌的分子机制，筛选LSM4、MYO1B变体的相互作用蛋白，阐明其调控机制，最终明确LSM4-MYO1B可变剪接信号促肝癌的关键机理。本研究将首次揭示LSM4在肝癌中的重要作用，阐明其促癌机制，为靶向RNA剪接体的小分子抑制剂研发提供理论依据。

Hepatocellular carcinoma (HCC) is an aggressive malignant tumor with the property of high lethality. Aberrant alternative splicing (AS) of RNA is tightly associated with the initiation and progression of HCC. However, currently it is urgently required to systemically identify and characterize the key AS factors-associated with the proliferation of HCC cells. In this study, the CRISPR-Cas9 technology was employed to screen for the essential AS factors for proliferation (ESF) in HCC cells. We found that a component from U6 snRNP spliceosome, the Sm-like protein 4 (LSM4), is potentially the most crucial ESF for HCC cells’ proliferation, and the LSM4-regulated-transcriptome analysis further indicated that LSM4 could modulate the AS of the 24th exon of MYO1B gene to play its oncogenic role. This project aims to investigate the expression, localization and biological function of LSM4 and its potential splicing variants – MYO1B-L and MYO1B-S using the models of HCC cells line, mice xenograft and HCC patients’ tissues. Through employing the methods of molecular molecular biology, transcriptomics and proteomics, we plan to: reveal the molecular mechanisms of LSM4 and MYO1B-L to promote HCC oncogenesis; screen and identify the interactors of LSM4 and the two MYO1B variants and to elucidate their regulatory mechanisms; finally confirm the crucial role of LSM4-MYO1B AS signalling pathway in HCC promotion. This study will, for the first time, reveal the important role of LSM4 in HCC, clarify its oncogenic mechanism, and provide a theoretical basis for the development of small molecule inhibitors targeting the RNA spliceosome.