肝癌Prp19相关复合物自身调控及其参与Wnt通路活化机制研究

Exotic acitivity of splicing factors contributes to the development of tumor. Splicing factors including pre-mRNA processing factor 19 (Prp19) and cell division cycle-like 5 (Cdc5L) within Prp19-associated complex were frequently upregulated in tissues of hepatocellular carcinoma (HCC) and were closely associated with the invasion and poor prognosis of HCC. Pilot study demonstrated that in HCC cell lines depletion of Prp19 inhibited Cdc5L expression rather than other core proteins within Prp19-associated complex. Positive correlation between Prp19 level and Cdc5L expression was observed in HCC tissue. In following work nuclear location sequence mutant, mass spectrometric analysis and phosphorylation mutant are used to elucidate the mechanism accounting for mutual regulation between Prp19 and Cdc5L. Data from cDNA microarray，real-time qPCR and immunoblot demonstrated that depletion of Prp19 impaired the activity of Wnt signal. The role of this complex on Wnt signal will be in depthe evaluated through RNA siliencing, transcriptome sequencing and transgenic mice model in the program. This work will address the link between dysregulation of splicing factors and development of HCC, sheding light on abnormal molecular events during HCC progressing and providing novel therapeutic targets of HCC.

剪接体异常参与肿瘤发生发展，Prp19相关复合物是重要的剪接体活化因子。课题组前期研究发现该复合物内核心蛋白Prp19与Ccd5L在多数肝癌中表达上调，二者表达呈正相关，与肝癌侵袭与预后不良密切相关；肝癌细胞系中下调Prp19抑制该复合物内Cdc5L而不是其他核心蛋白的表达；基因表达谱芯片、实时定量PCR、免疫印迹结果表明下调Prp19抑制Wnt信号的活性。本课题拟在上述工作的基础上，利用核定位序列突变体、质谱分析、磷酸化突变体等手段来阐明Prp19与Cdc5L间的相互调控机制；同时通过RNA干扰、转录组测序、基因敲除鼠模型来评估该复合物对Wnt信号通路的影响；希望能了解剪接因子异常与肝癌发展的关系，有助于阐明肝癌进展中异常分子事件，为肝癌治疗提供新靶点。

本研究发现肝细胞癌中Prp19复合物主要成分Prp19与Cdc5L间的调控及分子机制，并发现Prp19调控Wnt通路的下游主要靶点Cyclin D1的表达。体内外功能实验发现Prp19通过Cyclin D1参与肝癌细胞放化疗抵抗。后续机制研究阐明了Prp19通过WD40结构域与Cyclin D1结合，促进HCC的DNA损伤修复；Prp19通过调控eIF4E的表达，参与Cyclin D1的翻译，从而影响Cyclin D1的表达。在肝癌临床样本中Prp19和Cyclin D1的表达呈正相关，二者联合预测HCC患者的总体生存率和无病生存率比单一指标更具有应用价值。该研究有助于了解肝癌的生物学特性及放化疗抵抗相关分子机制，开发肝癌治疗增敏的靶点。