RNF2 Inhibits E-Cadherin Transcription To Promote Hepatocellular Carcinoma Metastasis Via Inducing Histone Mono-ubiquitination

Background: Hepatocellular carcinoma(HCC)is a common malignant tumor with high heterogeneity and high metastasis rate.More than 70% of patients are diagnosed in the middle and late stages due to metastasis,resulting in poor prognosis.RNF2 is a Pc G protein with E3 ubiquitin ligase activity,serving as a core catalytic element of PRC1,and participates in gene expression silencing by monoubiquitinating histone H2 A.RNF2 has been extensively studied as an oncogene in many types of tumors,but its role and specific mechanism in HCC are still unclear.Methods: First,the expression pattern of RNF2 in HCC was studied using bioinformatics analysis,Western Blot,and RT-qPCR quantification analysis in the TCGA-LIHC database,HCC cell lines,HCC clinical specimens,and HCC patient cohorts.The prognostic role of RNF2 in HCC and its correlation with clinical pathological features of HCC patients were also investigated.After confirming that RNF2 as an oncogene is likely associated with HCC metastasis,the role of RNF2 in promoting HCC invasion and migration was evaluated using Trans-well assays,wound healing assays,and nude mouse metastasis model experiments.It was preliminarily demonstrated that RNF2 may promote HCC metastasis by mediating EMT.ChIP experiments confirmed that RNF2 inhibits the transcription of Ecadherin by mediates histone modification,inducing EMT,and promoting HCC metastasis.Further exploration of the underlying reasons for the expression pattern of RNF2 in HCC.Transcription factors for RNF2 are predicted by analyzing and integrating the UCSC database and JASPAR database.Dual luciferase reporter gene assays and ChIP assays verified the role of the transcription factor NR2C2 in promoting the transcription of RNF2.Results: The results of bioinformatics analysis,Western Blot and RTqPCR quantification analysis of the TCGA-LIHC database,HCC cell lines,HCC clinical specimens,and HCC patient cohorts showed high expression of RNF2 in HCC cell lines and tissues,which was related to poor clinical pathological characteristics and prognosis of HCC patients.In vitro experiments showed that the invasive and migratory abilities of HCC cells were reduced with RNF2 knockdown,while RNF2 overexpression promoted these abilities.In addition,the results of WB and cellular IF assays showed that the expression of RNF2 was negatively correlated with the expression of epithelial markers(ZO-1,E-Cadherin and β-catenin)in EMT,and positively correlated with the expression of mesenchymal markers(N-Cadherin,Vimentin).In mechanism studies,the results of WB experiments showed that RNF2 overexpression promoted the expression of HAK119 ub and H3K27me3 while inhibiting the expression of H3K4me3,while RNF2 knockdown had the opposite effect.ChIP experiments showed that the overexpression of RNF2 promoted the enrichment of H2AK119 ub and H3K27me3 in the ECadherin promoter region while reducing the enrichment of H3K4me3;knockdown of RNF2 had the opposite effect.Meanwhile,we found that overexpression of RNF2 increased the protein stability of EZH2,the H3K27me3 methyltransferase,and reduced the protein stability of MLL1,the H3K4me3 methyltransferase.The results of in vitro experiments showed that the expression of E-Cadherin could reverse the effects of RNF2 expression on the invasive and migratory abilities of HCC cells and the expression of EMT-related proteins.In vivo experiments showed that overexpression of RNF2 promoted the metastasis of MHCC-LM3 cells in nude mice and the expression of tumor mesenchymal-related proteins,while inhibiting the expression of epithelial-related proteins.Consistent with the results of in vitro experiments,overexpression of E-Cadherin reversed the effects of RNF2 overexpression.Through analysis and integration of the UCSC database and JASPAR database,we identified six potential transcription factors for RNF2(HES7,HNF4 G,NR2C2,SP2,TCFL5,ZNF740).Through validation,we found that NR2C2 was highly expressed in HCC and directly affected the expression of RNF2,with its expression pattern positively correlated with RNF2.Dual luciferase reporter gene assays and ChIP assays showed that NR2C2 could bind to BS1 and BS2 in the promoter region of RNF2 and promote its transcription.Conclusion: The current study found that RNF2 played an oncogenic role in the progression of HCC by inducing EMT.RNF2 induced H2AK119 ub and cooperatively inhibited the transcription of E-Cadherin by crosstalk between H3K27me3 and H3K4me3,promoting the EMT process in HCC.Moreover,this study first revealed that NR2C2 could specifically bind to the BS1 and BS2 sites in the RNF2 promoter region,promoting the transcription of RNF2.These findings indicate that RNF2 promotes the metastasis of HCC by regulating the epigenetic modifications of the ECadherin promoter region and emphasize that RNF2 may be a potential prognostic marker and therapeutic target for HCC.