CLK1介导的可变剪接在细胞周期中的调控及其在胰腺癌进展转移机制中的研究

Pancreatic ductal adenocarcinoma (PDAC) was associated with poor prognosis. In recent years, increasing studies showed that alternative splicing (AS) events were strongly associated with cancer, and many oncogenes were regulated by AS events. However, the mechanism of AS events in pancreatic cancer is still unclear. It's reported that CLK1, one of SR protein kinases, was extensively involved in the regulation of AS events. Our preliminary study showed that CLK1 over-expression was positively correlated with PDAC grade and associated with worse prognosis. Also, disruption of CLK1 caused loss of proliferation and migration of pancreatic cancer cell. The level of CLK1 undergoes cell cycle dependent fluctuations, which reveals that CLK1 may play an important role in progression of PDAC by the means of CLK1-regulated periodic AS. However, there was no research of CLK1-regulated cell cycle-related AS on PDAC yet. Therefore, we firstly aim to manufacture samples at each phase of cell cycle under different CLK1 expression conditions using cell cycle synchronization. Secondly, we will use a cell model system of gene knock-out and whole transcription sequencing technology to explore the comprehensive regulatory network of CLK1 on periodic AS in PDAC. Moreover, we plan to identify the mechanism of CLK1-regulated periodic AS events, which was intimately associated with cancer cell cycle control and progression of pancreatic cancer. The present study may provide insights about the comprehensive networks of CLK1 on periodic AS events in PDAC. Meanwhile, it might provide a novel biomarker and treatment strategy in pancreatic cancer.

胰腺癌进展迅速、预后极差，RNA可变剪接在肿瘤进程中起着重要调控作用。我们通过敲除和过表达可变剪接蛋白激酶（CLK1），首先发现CLK1表达水平影响细胞周期并且与胰腺癌的恶性行为密切相关，但是CLK1调控细胞周期的机制研究近乎空白。本课题将聚焦于CLK1/剪接因子/细胞周期与胰腺癌的关系展开系列研究：1）利用已构建的敲除和过表达CLK1的胰腺癌细胞，通过激光共聚焦检测CLK1和剪接因子的合成、定位，结合流式细胞术等解释CLK1与细胞周期的关系；2）转录组测序获得细胞周期差异表达谱，原位杂交技术确认剪接片段；3）通过WB和Co-IP验证差异表达谱，筛查CKL1的活化因子，探索CKL1在细胞周期中的剪接调控机制。课题将验证CKL1可变剪接调控细胞周期推进肿瘤进展的假说，为胰腺癌新靶点的挖掘奠定基础。

胰腺癌被称为“癌中之王”，胰腺癌的五年生存率低依然徘徊于5%左右。胰腺癌因缺乏早期特异性症状，发病过程极为隐匿，且拥有极强的侵袭性，大部分患者在首次诊断时就已为晚期，错失手术机会，同时，胰腺癌对放疗以及化学治疗均不敏感，治疗手段极度匮乏。目前关于胰腺癌的发病原因，发展机制依旧不甚明了，深入探索胰腺癌发生发展中的表观遗传学事件，探索全新胰腺癌分子标记物以及治疗靶点具有重要的临床意义以及社会价值。CLK1是介导可变剪接的重要功能分子,已被报道与多种恶性肿瘤密切相关，然而，在胰腺癌中，CLK1的表达情况以及作用国内外均未见报道。本研究首次明确CLK1在胰腺癌中高表达且与胰腺癌患者预后密切相关。且通过体内外的分子生物学实验研究CLK1与胰腺癌的细胞周期紊乱及增殖密切相关。我们对CLK1下游可能调控的剪接因子进行筛选并验证其功能，并完整的观测了CLK1下游介导的差异可变剪接事件。本研究旨在阐明CLK1在胰腺癌细胞周期紊乱的作用及其机制，对CLK1在胰腺癌中的具体作用机制进行完整探索讨论，力图对胰腺癌早期诊断以及靶向治疗提供新的科学基础以及理论依据。