Insulin Degrading Enzyme Mediates Resistance To Sorafenib In Hepatocellular Carcinoma By Inhibiting AR Ubiquitination

Background:Hepatocellular carcinoma(HCC)is a common malignant tumor of the digestive system.Sorafenib is a first-line drug for the treatment of advanced HCC.Although Sorafenib has a good therapeutic effect on HCC,there is a problem that tumors are prone to drug resistance.Exploring the molecular mechanism of drug resistance in HCC may contribute to solve the common clinical problem.Methods:We established Sorafenib-resistant HCC cell lines and used these cell lines to perform RNA sequencing.We also searched the GEO and TCGA databases,used bioinformatics analysis to screen the potential key genes related to Sorafenib resistance.The real-time quantitative PCR(q PCR),Western Blot(WB)and immunohistochemical staining were used to verify the expression level of the gene.Techniques of gene overexpression and CRISPR-CAS9 knockout were used to increase and decrease gene expression.The enzyme inhibitor(Bacitracin)was used to inhibit the protein function.Cell Counting Kit-8(CCK8),clone formation,cell cycle assay,in vivo animal exprienment were used to observe the gene function on the process of Sorafenib resistance.Moreover,we explored the underlying role of the gene in Sorafenib resistance by using the bioinformatics analysis,q PCR experiment,WB experiment,cell immunofluorescence staining,immunoprecipitation,protein degradation detection,protein ubiquitination level detection and rescue experiments.Results:Bioinformatics analysis identified that the expression of insulin degrading enzyme(IDE)significantly increased in the Sorafenib-resistant cell lines,and it was also associated with the poor prognosis of HCC.The q PCR and WB analysis verified that the expression of IDE increased in Sorafenib-resistant cells.The survival analysis of patients with HCC undergoing Sorafenib treatment also showed that elevated IDE was associated with poor prognosis.Overexpression of IDE enhanced cell proliferation and impaired sensitivity to Sorafenib.Knocking out IDE or inhibition of IDE function inhibited cell proliferation and enhanced sensitivity to Sorafenib.In vivo,the tumor formation of IDE knockout group was inhibited and the combined use of Sorafenib and bacitracin enhanced the therapeutic effect of Sorafenib.Bioinformatics analysis revealed that IDE was closely related to AR signaling pathway.Immunofluorescence staining and immunoprecipitation revealed that IDE binds to androgen receptor(AR).By detecting the ubiquitination and degradation of AR,it was found that IDE inhibited the degradation of AR through the ubiquitination pathway,resulting in enhancement of the AR downstream signal pathway,and finally mediating the process of Sorafenib resistance.Conclusion:In this study,high-throughput gene sequencing,cell experiments and in vivo animal experiments found that the increased IDE was closely related to Sorafenib resistance.Through bioinformatics analysis,protein interaction analysis and rescue experiments,we found that IDE inhibited AR degradation through the ubiquitination-proteasome pathway,and then regulated AR downstream signaling pathways and mediated the process of Sorafenib resistance...