环指蛋白RNF125调控抑癌基因RIG-I与TUSC2相关蛋白泛素化降解参与肝癌进展的机制研究

The dysfunction of ubiquitin modification has been demonstrated to be closely related to tumor development, and a variety of ubiquitin ligases were confirmed to be oncogene or tumor suppressor genes. We previously reported E3 ubiquitin ligases UBE3C and deubiquitinase USP7 were associated with the invasion and growth of hepatocellular carcinoma (HCC, Hepatology, 2013 and 2014). Recently, we studied the impact of the tetraspanin-enriched microdomains (TEMs) on the TNFα/TNFR1 function. With the analysis of SILAC, we detected the ring finger protein 125 (RNF125), an E3 ubiquitin ligases, was significantly up-regulated in HCC cells treated by TNFα. Furthermore, the Co-immunoprecipitation combined with mass spectrometry showed the tumor suppressor genes RIG-I and TUSC2 related protein were the substrate of RNF125. Thus, we hypothesized that high expression of RNF125 promoted HCC progression, and there might be another important new approach, ubiquitin degrading RIG-I and TUSC2 protein, for the HCC progression induced by TNFα. In the proposal, we will assess the expression of RNF125, RIG-I and TUSC2 expression in hepatocellular carcinoma tissues, and investigate the correlations among the RNF125, RIG-I and TUSC2 expression, and the relationship between their expression with the clinicopathological characteristics and prognosis of HCC patients. By regulating the expression of RNF125, we investigated the biological functions of RNF125 in HCC cells. Then, the regulation and mechanisms of RNF125 on RIG-I and TUSC2 protein was further addressed. Finally, the biological functions and mechanism of RNF125 in HCC cells were further confirmed in vivo. Our study aims to further elaborate the role of ubiquitin in HCC, and possibly develop a novel therapeutic target for HCC.

泛素化修饰异常与肿瘤进展密切相关，目前多种泛素连接酶被证实为癌或抑癌基因。我们既往报道泛素连接酶UBE3C与去泛素化酶USP7高表达参与肝癌侵袭与生长（Hepatology，2014与2015）。近期研究四跨膜网络对TNFα/TNFR1功能影响，用SILAC分析TNFα诱导肝癌细胞蛋白的表达，发现泛素连接酶RNF125在TNFα处理细胞显著上调，免疫共沉淀联合质谱示抑癌基因RIG-I与TUSC2相关蛋白为其作用底物。推测RNF125高表达促进肝癌进展，其泛素化降解后两者可能是TNFα诱导肝癌进展的另一重要新途径。故拟在组织中研究RNF125、RIG-I及TUSC2表达，分析三者相关性及与肝癌临床病理和患者预后关系；细胞中调节RNF125表达研究其对肝癌细胞生物学功能影响；分子水平研究其对RIG-I与TUSC2作用机制并体内验证。课题将进一步阐述泛素化修饰在肝癌中作用，为肝癌治疗提供新靶点。

近年，泛素化修饰异常（特别是E3泛素链接酶）在肝癌发生发展中发挥重要作用。我们前期发现肿瘤坏死因子TNFα作用于肝癌细胞后，E3泛素链接酶RNF125表达上调。本课题在前期工作基础上，证实：（1）RNF125在大部分肝癌组织中表达高于相应癌旁组织，且与肝癌患者恶性临床病理特征及预后与复发相关。（2）在肝癌细胞中调节RNF125表达发现，高表达RNF125促进肝癌侵袭、移动及增值。且体内实验也发现高表达RNF125促进肝癌进展。（3）通过免疫共沉淀结合质谱分析证实明确抑癌基因RIG-I与TUSC2蛋白可能是RNF125作用底物；通过构建RNF125、RIG-I与TUSC2标签质粒结合泛素化相关实验证实RNF125对RIG-I与TUSC2蛋白的泛素化降解作用。（4）体内实验证实抑制RNF125表达能阻断TNFα诱导的肝癌进展。（5）临床上高RNF125，低RIG-I与TUSC2蛋白表达患者预后较单独表达者差。另外本课题揭示E3泛素链接酶RNF38高表达通过泛素化降解AHANK1易化TGF-β信号参与肝癌进展。本研究通过体内外实验阐述RNF125与RNF381参与肝癌进展及其机制，为RNF125与RNF38作为肝癌侵袭转移的治疗靶点提供新的理论依据。