FAT10调控WISP1表达在肝癌细胞增殖中的作用及机制研究

Our study had demonstrated that FAT10 was a special ubiquitin-like protein which could both stabilize and degrade the substrate protein, and was highly expression and promoted cell proliferation in hepatocellular carcinoma (HCC) (Cancer Research 2016).Besides, it has been proved that Wnt-1 induced secreted protein-1(WISP1) is intimately related to tumor proliferation. However, the relationship between FAT10 and WISP1 still remains unclear. Through a series of assays including quantitative proteomics and immunohistochemistry, we found that WISP1 was low expressed in HCC and was negatively correlated with FAT10 expression. Overexpression of WISP1 could suppress HCC proliferation. Down-regulation of FAT10 could increase the expression of WISP1 and then inhibit the proliferation of hepatocellular carcinoma cells. Co-immunoprecipitation (Co-IP) results confirmed a mutual interaction between FAT10 and WISP1. Therefore, we speculated that FAT10 might promote the proliferation of hepatocellular carcinoma cells via directly degrading WISP1. To verify this hypothesis, we intend to perform further experiment. Firstly, we confirm the role of FAT10 regulating WISP1 in the proliferation of hepatocellular carcinoma cells by in vivo and in vitro experiments. Secondly, through the experiment of site-directed mutagenesis and in vitro ubiquition, the mechanism of WISP1 regulation by FAT10 will be discussed. Finally, the expression of FAT10 and WISP1 in HCC tissues will be detected to analyze the correlation between them and the correlation with clinical pathology and prognosis.This study will provide a new theoretical foundation for prevention and treatment of hepatocellular carcinoma proliferation.

我们研究证实FAT10是一种既能稳定又可降解底物的类泛素蛋白，其在肝癌中高表达并促进肝癌细胞增殖（Cancer Research 2016）。另外，研究表明Wnt诱导分泌蛋白WISP1与肿瘤增殖密切相关。但两者关系尚不清楚。前期我们通过定量蛋白质组学及免疫组化等实验发现肝癌组织中WISP1呈低表达，且与FAT10表达呈负相关；过表达WISP1可抑制肝癌细胞增殖；降低FAT10表达可导致WISP1表达增加并抑制肝癌细胞增殖，同时免疫共沉淀提示两者相互结合。据此我们推测FAT10通过直接降解WISP1促进肝癌细胞增殖。为验证假说，本课题①通过体内外实验明确FAT10调控WISP1表达对肝癌细胞增殖的影响，②通过点突变、体外泛素化等实验探讨FAT10调控WISP1的机制，③检测临床肝癌组织中FAT10和WISP1表达，分析两者相关性及与临床病理和预后的关联，从而为肝癌细胞增殖的防治提供理论依据。

我们研究证实FAT10是一种既能稳定又可降解底物的类泛素蛋白，其在肝癌中高表达并促进肝癌细胞增殖。另外，研究表明Wnt诱导分泌蛋白WISP1与肿瘤 增殖密切相关。但两者关系尚不清楚。在这项研究中，首先证实WISP1在肝细胞癌组织中显著下调，是HCC病人不良预后的一个独立预测因子，并且体内和体外实验的证据均表明WISP1能够抑制HCC细胞的增殖。更进一步的研究发现WISP1的低表达与FAT10的表达有关，FAT10是一个泛素样蛋白，既可以发挥促进蛋白降解的功能也可以起到稳定作用，是参与蛋白翻译后修饰的一个重要蛋白。我们发现FAT10的过表达能够通过FAT10修饰（FAT10ylation）促进WISP1降解进而降低WISP1表达促进HCC细胞增殖。有趣的是，FAT10过表达会导致WISP1发生蛋白/mRNA表达的不一致，出现WISP1蛋白水平下降而mRNA水平增加的情况。机制探索发现：FAT10能够同时发挥促进底物稳定和促进底物降解的功能，FAT10过表达会通过稳定beta-catenin促进WISP1 mRNA表达，并直接促进WISP1蛋白发生降解。总的来说，这些结果表明FAT10过表达会导致WISP1出现mRNA和蛋白表达的不一致，因此通过下调WISP1的蛋白水平促进HCC进展。