E3泛素化连接酶RNF14介导肝细胞核因子HNF1α泛素化降解促进肝癌增殖及转移的机制研究

Hepatocyte nuclear factor 1α (HNF1α) is a suppressor of HCC proliferation and metastasis. Our previous studies showed that HNF1α protein level was lower in HCC tissue compared with the noncancerous tissues, but HNF1α mRNA level showed no significant difference, while the mechanism remain unclear. Abnormal ubiquitin-degradation of tumor-suppressor gene is the important reason for the development of HCC. Database analysis results demonstrated E3 ubiquitin ligase RNF14 directly combined with HNF1α, and our results revealed that RNF14 promoted HNF1α degradation by the proteasome system. Further studies showed that RNF14 was overexpressed in HCC tissue and acted as oncogene to promote the proliferation and migration of HCC cells. Therefore, we hypothesized that RNF14 promotes HCC proliferation and metastasis via inducing HNF1α degradation mediated by ubiquitin-proteasome system (UPS). We intend to detect RNF14 and HNF1α expression in human HCC tissue, then analyzed the relationship between RNF14 expression with clinical malignant phenotype and prognosis of HCC. Moreover, we will demonstrated the important role and mechanism of RNF14 in promoting HCC proliferation and metastasis through inducing HNF1α degradation mediated by UPS in HCC cells and a variety of HCC animal models. In this study, we try to clarify the mechanism of HNF1α reduced in HCC tissue and the pivotal role of RNF14 in promoting proliferation and metastasis of HCC, which will provide new strategy for clinical diagnosis and treatment of HCC.

肝细胞核因子HNF1α是肝癌增殖和转移的抑制因子，前期研究发现肝癌组织中HNF1α蛋白水平较癌旁组织明显降低，但是HNF1α mRNA水平却无明显差异，机制不明。抑癌基因异常泛素化降解是肝癌发生发展的重要原因。数据库分析结果显示E3泛素化连接酶RNF14与HNF1α存在直接结合，预实验结果也证实RNF14可促进HNF1α经蛋白酶体系统降解，且RNF14在肝癌组织中高表达，可促进肝癌细胞增殖及迁移。因此，提出假设：RNF14通过介导HNF1α泛素化降解促进肝癌增殖及转移。本课题拟检测人肝癌组织中RNF14与HNF1α表达相关性，并探讨其与肝癌临床恶性表型和预后的关系；利用肝癌细胞及多种动物模型，明确RNF14通过介导HNF1α泛素化降解进而促进肝癌增殖及转移的作用及作用机制。以期阐明肝癌组织中HNF1α表达降低机制以及RNF14在肝癌增殖及转移中的重要作用，为肝癌临床诊治提供新思路和方法。

肝细胞核因子HNF1α是肝癌增殖和转移的抑制因子，HNF1α在肝癌组织中表达降低，但是其mRNA水平却无明显差异，机制不明。本研究发现：1.RNF14在肝癌组织和肝癌细胞中的表达分别显著高于癌旁组织和正常肝细胞(P<0.05)；2.Transwell侵袭迁移实验结果显示:过表达RNF14后,肝癌细胞Hu7的迁移与侵袭能力显著高于阴性对照组(P<0.05),同样地,敲低RNF14后,肝癌细胞7404的迁移与侵袭能力显著低于阴性对照组(P<0.05)。3.CCK-8增殖实验、克隆形成与皮下移植瘤实验结果证明:敲低RNF14能够显著抑制肝癌细胞的增殖(P<0.05)。4.免疫共沉淀实验结果显示:RNF14与HNF1α之间存在相互结合,且RNF14能够下调HNF1α的表达。综上所述，本研究发现RNF14能够通过抑制抑癌因子HNF1α的表达,增强肝癌细胞的增殖与侵袭转移能力,从而在肝癌的发生、发展中起到重要作用。这些创新性发现进一步阐明肝癌发生发展机制并提供潜在诊治靶点，具有潜在的临床转化价值。