SRPK1通过BCLAF1介导调控肝癌细胞抵抗凋亡的机制研究

Aberrant regulation of cell apoptosis is one of important reasons for tumor development. The splicing factor kinase SRPK1, which plays a crucial role in the regulation of cell apoptosis and contributes to tumorigenesis. However, the underlying mechanisms by which SRPK1 is involved in cell apoptosis are still to be classified. In search of SRPK1-interacting proteins by CoIP and Mass Spectrum analysis, we identified an apoptosis-related protein BCLAF1 (Bcl-2 associated transcription factor 1) as a potential downstream target of SRPK1. Interestingly, BCLAF1 could interact with SRPK1 in vivo and in vitro. The overexpression of SRPK1 could phosphorylate BCLAF1 and promote BCLAF1 translocation from nucleus to cytoplasm. Functionally, BCLAF1 knockdown by specific small RNA inference attenuated the resistance of SRPK1 overespressing cells to apoptosis. Taken together, these results suggest that SRPK1 controls cell apoptosis by BCLAF1 in liver cancer cells. In this project, we will apply to CoIP analysis and provide molecular basis of the interaction betweeen SRPK1 and BCLAF1 by constructing various truncated mutants, further investigate the effect of BCLAF1 subcellular distribution and phosphorylation level on Bcl-2 apoptotic signal transduction, apoptosis-related gene transcription and some mRNA alternative splicings of DNA repair factors and finally explore the functional relevance of BCLAF1 and SRPK1 proteins in liver clinical samples. Our project will illuminate the molecular mechanism by which SRPK1 modulates apoptosis through BCLAF1 and provide new potential targets for tumor therapy and drug resistance.

细胞凋亡调控异常是肿瘤发生的一个重要原因，剪接因子激酶SRPK1已被证实促进多种肿瘤发生并抑制细胞凋亡，但其抑制凋亡机制不明。预实验发现SRPK1能够结合并磷酸化其潜在底物BCLAF1（Bcl-2相关凋亡调控因子），过表达SRPK1可上调BCLAF1蛋白水平并使其发生核质转位，功能上下调BCLAF1表达能够减弱SRPK1过表达细胞抵抗化疗药物诱导的凋亡。据此推测SRPK1通过BCLAF1介导抑制细胞凋亡发生。本课题拟通过构建一系列突变体分析BCLAF1与SRPK1的相互作用和验证SRPK1对BCLAF1磷酸化及核质转位的影响；运用上位表达实验研究SRPK1/BCLAF1在细胞凋亡中的上下游关系；通过临床样本分析二者在肝癌发生中的临床意义及关联，最终揭示BCLAF1下游的凋亡通路或效应因子，阐明BCLAF1介导SRPK1调控细胞凋亡的分子机制，为肿瘤发生和化疗耐药提供新的靶向药物研发依据。

剪接因子激酶SRPK1已被证实促进多种肿瘤发生并抑制细胞凋亡，但其抑制凋亡机制不明。我们前期报道了SRPK1在肝癌发生中的作用及部分分子机制，在本项目中我们揭示了一个全新的SRPK1/BCLAF1信号轴线，阐明了其参与肝癌发生并促进肝癌细胞抵抗凋亡的作用和机制。免疫共沉淀实验表明SRPK1与BCLAF1能够发生相互结合，质谱分析SRPK1能够磷酸化BCLAF1蛋白N端区域S25和S148，过表达SRPK1可上调BCLAF1蛋白水平并使其发生核质转位，而N端缺失的BCLAF1截短体蛋白则不受该调控。功能上，下调BCLAF1表达能够减弱SRPK1过表达细胞抵抗化疗药物诱导的凋亡；裸鼠水平皮下成瘤模型中，BCLAF1下调同样明显减弱SRPK1过表达细胞的致瘤能力。进一步对BCLAF1下游功能和调控机制的探索发现，BCLAF1通过诱导自噬促进肝癌细胞在药物应激条件下的存活；RNAseq测序结果还发现了多个受到SRPK1和BCLAF1共同调控的致癌基因包括HMHA2，STC2，NYCN和S100A9等，这些因子可能通过除自噬外的其他机制介导SRPK1和BCLAF1对细胞凋亡的调控。在对肝癌样本的免疫组化分析中我们发现SRPK1和BCLAF1在癌组织中表达显著升高，而二者表达存在明显的正相关；通过对临床病理参数和预后的分析发现BCLAF1高表达与高TNM分期有统计学意义，同时患者生存期较差。这些结果表明了BCLAF1受到SRPK1调控，在功能上通过调控细胞自噬介导SRPK1抵抗细胞凋亡，为肿瘤发生和化疗耐药提供了新的靶向药物研发依据。