新型E3泛素连接酶对肝癌细胞抵抗失巢凋亡的作用效应和分子机制

Ubiquitination, a protein modification pattern with high specificity, regulate multiple cellular pathophysiology activities in cells. E3 ubiquitin ligase has a critical role of identifying target protein in this process. Tripartite motif 31 (TRIM31) is a recently discovered E3 ubiquitin ligase family member. The expression and function of TRIM31 in hepatocellular carcinoma has not yet reported.Tripartite motif 31 (TRIM31) is one of the new functional proteins with E3 ubiquitin ligase activity. However, the expression and function of TRIM31 in hepatocellular carcinoma (HCC) has not been reported. Our previous analysis in clinical specimens firstly showed that TRIM31 is up-regulated in HCC, and its overexpression is positively correlated with tumor metastasis. The in vitro transfecting experiments further confirmed that TRIM31 may promote the metastasis and invasion activity of tumor cells. Anoikis experiment using HCC cells showed that, the expression level of TRIM31 is significantly elevated, while silencing TRIM31 may reverse its enhanced ability of invasion and resisting anoikis. The preliminary results indicates TRIM31 is involved in HCC anoikis and therefore promote tumor invasion and metastasis. Based on these results, we have planned the further study on exposing the role of TRIM31 in HCC in three levels: cell model, animal model and patients clinical features, which also constructed a multi-dimensional research system to explore the potential acting mechanisms of anoikis. This study may provide effective targeted therapies with the theoretical supports.

蛋白质的泛素化修饰具有高度特异性，它参与调节细胞内多种病理生理活动。其中E3泛素连接酶对靶蛋白特异性识别起关键作用。Tripartite motif 31 (TRIM31)是近年发现的一种新型E3泛素连接酶，其功能尚未完全阐明，而在肝癌中表达及功能未见报道。申请人前期工作中首次发现TRIM31高表达于肝癌组织中，且与肝癌侵袭转移正相关，进一步对其过表达和沉默，证实TRIM31促肿瘤侵袭转移作用。肝癌细胞失巢试验显示，失巢后肝癌细胞TRIM31表达显著升高，而对其沉默后，逆转了肝癌细胞抵抗失巢凋亡和侵袭力。因此TRIM31可能介导肝癌细胞抗失巢凋亡而促进侵袭转移。本课题拟以此为切入点，通过细胞实验、动物实验和临床资料分析，明确TRIM31在肝癌抗失巢凋亡中的作用及机制。此研究提供了一个从分子水平、细胞水平到动物水平的研究体系，为深入了解肝癌抗失巢凋亡机制及寻找肝癌侵袭转移新调控靶点提供依据。

Tripartite motif 31 (TRIM31)是近年发现的一种新型E3泛素连接酶，其功能尚未完全阐明，而在肝癌中表达及功能未见报道。本课题在前期工作中首次发现TRIM31高表达于肝癌组织中，且与肝癌侵袭转移正相关，进一步对其过表达和沉默，证实TRIM31促肿瘤侵袭转移作用。肝癌细胞失巢试验显示，失巢后肝癌细胞TRIM31表达显著升高，而对其沉默后，逆转了肝癌细胞抵抗失巢凋亡和侵袭力。因此TRIM31参与介导肝癌细胞抗失巢凋亡而促进侵袭转移。在此前期研究基础上，本研究首先再次对TRIM31参与肝癌细胞抵抗失巢凋亡的作用进行验证。在下面的分子机制的研究中，我们过表达TRIM31后，发现对p53表达有负向调控作用，其中AMPK信号通路的激活介导了肝癌细胞的抵抗失巢凋亡效应。为了进一步研究TRIM31与p53的作用模式，我们过表达TRIM31和p53，应用免疫共沉淀试验验证实TRIM31可以和内/外源性p53直接靶向结合；在外源性共转染TRIM31和Ub后，免疫共沉淀试验证实TRIM31靶向p53进行泛素化修饰降解，从而激活AMPK信号通路提高肝癌细胞抵抗失巢凋亡能力。另外，为了精确了解TRIM31发挥功能的关键结构域，我们设计了RING结构域缺失和突变的表达载体转染肝癌细胞，明确了 RING结构域是其发挥泛素化修饰功能的关键功能区域，同时p53的K48位点是其泛素化位点。本研究对TRIM31介导肝癌细胞抵抗失巢凋亡进一步侵袭转移作用的机制进行详细的研究阐明，其研究结果将为针对肝癌转移的药物研发提供了新的思路和理论。