Study On The Roles And Mechanism Of RBCK1 Stabilizing RNF31 In Hepatocellular Carcinoma Progression

Background: hepatocellular carcinoma(HCC)exhibited a highly malignant biological behavior that caused a large number of tumor-related deaths.Primary liver cancer is not sensitive to chemotherapy,and surgical treatment for advanced primary liver cancer is poor.The LUBAC complex consists of RNF31,RBCK1 and SHARPIN.Previous studies have shown that the LUBAC contributes to the regulation of numerous physiological and pathological processes,such as inflammation.In recent years,some scholars have found that LUBAC also plays a critical part in developing tumors.Previously reported that SHARPIN facilitates HCC development,however,to our knowledge,the function and related molecular mechanisms of RBCK1 and RNF31 in HCC have not been investigated.Objective: By studying the biological function and interaction mechanism of RNF31 and RBCK1 in hepatocellular carcinoma and the correlation between the expression of RNF31 and RBCK1 and clinical data of HCC patients,we hope this work will provide new targets for the diagnosis and treatment of hepatocellular carcinoma in the clinic.Methods:(1)The expression of RNF31 and RBCK1 in non-tumor tissues and tissues of HCC pathological specimens were detected by TCGA online database analysis and protein immunoblotting technique and immunohistochemical experiment in our sample bank.(2)The relationship between RNF31 and RBCK1 expression and prognosis in HCC was analyzed by TCGA online database and immunohistochemical experiment in our sample bank.(3)The Fisher test was used to analyze the correlation between protein expression and clinical characteristics of RNF31 and RBCK1 in HCC.(4)q PCR detected the m RNA expression of RNF31 in HCC cell lines,and western-blot detected the protein expression of RNF31 and RBCK1 in HCC cell lines.(5)RNF31 and RBCK1 knockdown HCC cell lines were constructed,and the effects of knockdown RNF31 and RBCK1 on the oncogenic role of HCC were determined in vitro.(6)Lung tumor metastasis in mouse models was established by tail vein injection of control and RNF31-silenced huh-7 cells.Huh-7cells with or without RNF31-knockdown were subcutaneously inoculated into the right and left flanks of nude mice.(7)HCC cells were then incubated with varying concentrations of gliotoxin.(8)The m RNA expression level of RNF31 in HCC cell lines after knockdown of RBCK1 was determined via q PCR,and western-blot was used to detect the protein expression level of RNF31 in HCC cell lines after knockdown of RBCK1.(9)RNF31 protein level after the knockdown of RBCK1 in PLC/PRF/5 and huh-7 cells was analyzed by western blot.(10)Immunofluorescence co-staining of RBCK1 and RNF31 in wild-type PLC/PRF/5 and huh-7 cells to detect colocalization.Co-immunoprecipitation of endogenous RBCK1 and RNF31 in wildtype PLC/PRF/5 and huh-7 cells.(11)The effect of knockdown of RBCK1 on the stability of RNF31 was investigated by protein half-life assay.(12)The control and RBCK1 knockdown PLC/PRF/5 and huh-7 cells were pre-treated with the DMSO,lysosome inhibitor,chloroquine,or the proteasome inhibitor,MG132,and then RBCK1,RNF31,and β-actin were detected via western blot.The control and RBCK1 knockdown PLC/PRF/5 and huh-7 cells were incubated with MG132.The cell lysates were immunoprecipitated with anti-RNF31 antibody and then immunoblotted with the indicated antibodies.(13)Rescue experiment was used to verify RNF31 regulation by RBCK1.(14)Immunohistochemistry was used to score RBCK1 expression in HCC,and Spearman correlation analysis was applied to examine the correlation between RNF31 and RBCK1 immunohistochemistry scores.(15)Through exogenous immunoprecipitation mass spectrometry of RNF31,quantitative protein and quantitative protein ubiquitin mass spectrometry before and after knockdown of RNF31,the downstream molecules of RNF31 were initially explored.Results:(1)RNF31 and RBCK1 were highly expressed in HCC tissues as verified by TCGA online database analysis as well as protein immunoblotting technique and immunohistochemical scoring in the pathology specimen library.(2)High expression of RNF31 and RBCK1 in HCC was correlated with poor prognosis by analyzing the TCGA online database and immunohistochemical scores.(3)The Fisher test analysis analyzed that the IHC score of RNF31 and clinical features of metastasis were significantly correlated.(4)RNF31 and RBCK1 knockdown HCC cell lines were constructed,and in vitro experiments confirmed that knockdown of RNF31 and RBCK1 could inhibit HCC invasion,migration,and proliferation.(5)Subcutaneous tumor formation in nude mice confirmed that knockdown of RNF31 could inhibit the proliferation of HCC cells.The study of the mouse tail vein lung metastasis model confirmed that knockdown of RNF31 could inhibit the migration and invasion of HCC cells.(6)The RNF31 inhibitor gliotoxin inhibits the malignant behavior of HCC cells.(7)Immunofluorescence analysis showed the colocalization of RBCK1 and RNF31,and a co-immunoprecipitation assay experiment confirmed the RBCK1/RNF31 interaction in HCC cells.(8)After the knockdown of RBCK1,the m RNA of RNF31 did not change,but the protein level decreased.(9)The protein of RBCK1 did not change after the knockdown of RNF31.(10)The protein half-life experiments proved that RBCK1 positively regulates the stability of RNF31.(11)Partial regulation of RNF31 by RBCK1 through the ubiquitin-proteasome pathway was demonstrated by adding MG132 drug and CQ drug and ubiquitination experiments.(12)The rescued experiments verified that overexpression of RNF31 could partially revert the effect of knockdown of RBCK1 in HCC.(13)Spearman correlation analysis showed a significant positive correlation between RNF31 and RBCK1 immunohistochemical scores.Conclusion: Here,we investigated the role of RNF31 and RBCK1 in HCC.We showed that RNF31 and RBCK1 were overexpressed in HCC,and that upregulation of RNF31 and RBCK1 indicated poor clinical outcomes in patients with HCC.RNF31 overexpression resulted in more satellite foci and vascular invasion in patients with HCC.Additionally,RBCK1 expression correlated positively with RNF31 expression in HCC tissues.Functionally,RBCK1 and RNF31 promote the metastasis and growth of HCC cells.Moreover,the RNF31 inhibitor gliotoxin inhibited the malignant behavior of HCC cells.Mechanistically,RBCK1 interacted with RNF31 and repressed its ubiquitination and proteasomal degradation.In summary,the present study revealed an oncogenic role and regulatory relationship between RBCK1 and RNF31 in facilitating proliferation and metastasis in HCC,suggesting that they are potential prognostic markers and therapeutic targets for HCC.