DataSheet3_HIF-1/2α-Activated RNF146 Enhances the Proliferation and Glycolysis of Hepatocellular Carcinoma Cells via the PTEN/AKT/mTOR Pathway.xlsx

Hypoxia microenvironment, a critical feature of hepatocellular carcinoma, contributes to hepatocarcinogenesis, tumor progression and therapeutic resistance. Hypoxia-inducible factors (HIFs)-activated target genes are the main effectors in hypoxia-induced HCC progression. In this study, we identified ubiquitin E3 ligase ring finger protein 146 (RNF146) as a novel HIFs target gene. Either HIF-1α or HIF-2α knockdown significantly repressed hypoxia-induced RNF146 upregulation in Hep3B and Huh7 cells. TCGA data and our immunohistochemistry analysis consistently revealed the overexpression of RNF146 in HCC tissues. The upregulated expression of RNF146 was also detected in HCC cell lines. The high RNF146 level was correlated with poor clinical features and predicted a shorter overall survival of patients with HCC. RNF146 knockdown suppressed the proliferation, colony formation and glycolysis of HCC cells, but suppressed but RNF146 overexpression promoted these malignant behaviors. Moreover, RNF146 silencing weakened HCC growth in mice. RNF146 inversely regulated phosphatase and tensin homolog (PTEN) protein level, thereby activating the AKT/mechanistic target of rapamycin kinase (mTOR) pathway in HCC cells. MG132 reversed RNF146 overexpression-induced PTEN reduction. RNF146 knockdown decreased the ubiquitination and degradation of PTEN in HCC cells. Therefore, we clarified that PTEN knockdown notably abolished the effects of RNF146 silencing on the AKT/mTOR pathway and Hep3B cells’ proliferation, colony formation and glycolysis. To conclude, our data confirmed that RNF146 was transcriptionally regulated by HIF-1/2α and activated the AKT/mTOR pathway by promoting the ubiquitin proteolysis of PTEN, thereby contributing to HCC progression. RNF146 may be a potential new drug target for anti-HCC.

缺氧微环境是肝细胞癌（hepatocellular carcinoma, HCC）的关键特征之一，其参与肝细胞癌变、肿瘤进展及治疗耐药的发生过程。缺氧诱导因子（hypoxia-inducible factors, HIFs）激活的靶基因是缺氧诱导肝癌进展的主要效应分子。本研究中，我们将泛素E3连接酶环指蛋白146（RNF146）鉴定为一种新型HIFs靶基因。敲低缺氧诱导因子-1α（HIF-1α）或缺氧诱导因子-2α（HIF-2α），均可显著抑制缺氧诱导的Hep3B与Huh7细胞中RNF146的上调表达。TCGA数据库数据与本研究的免疫组化分析均一致显示，RNF146在肝癌组织中呈高表达。肝癌细胞系中同样检测到RNF146的上调表达。RNF146高表达与肝癌患者不良临床特征相关，且可预测患者更短的总生存期。敲低RNF146可抑制肝癌细胞的增殖、集落形成与糖酵解过程，而过表达RNF146则可增强上述恶性生物学行为。此外，敲低RNF146可抑制小鼠体内肝癌的生长。RNF146可负向调控张力蛋白同源磷酸酶基因（phosphatase and tensin homolog, PTEN）的蛋白水平，进而激活肝癌细胞中的AKT/雷帕霉素靶蛋白激酶（mTOR）信号通路。蛋白酶体抑制剂MG132可逆转RNF146过表达所诱导的PTEN蛋白水平下降。敲低RNF146可降低肝癌细胞中PTEN的泛素化修饰与降解过程。因此，我们证实：敲低PTEN可显著抵消RNF146沉默对AKT/mTOR通路，以及对Hep3B细胞增殖、集落形成与糖酵解的影响。综上，本研究数据证实：RNF146受HIF-1/2α的转录调控，并通过促进PTEN的泛素蛋白酶体降解激活AKT/mTOR通路，进而参与肝癌进展。RNF146有望成为抗肝细胞癌的新型潜在药物靶点。