Inhibitory Effect And Mechanism Of Ribavirin On Hepatocellular Carcinoma Via Mediating CARM1 And PRMT5

Background: Reversible methylation of histone and other proteins is a key posttranslational modification process,which is related to cell development and tumorigenesis.In mammalian cells,nine protein arginine methyltransferases(PRMTs)contribute to catalyze arginine methylation.Coactivator-associated arginine methyltransferase 1(CARM1)ia a type I PRMTs that catalyze the asymmetric dimethylation of arginine residues on protein substrates.Protein arginine methyltransferase 5(PRMT5)is a type II PRMTs that catalyze the symmetric dimethylation of arginine residues on protein substrates.CARM1 and PRMT5 are frequently overexpressed in cancer cells,and their elevated expression correlates with poor prognosis,thus making them excellent therapeutic targets.Selective smallmolecule inhibitors of CARM1 or PRMT5 have been generated.Ribavirin(Rib),as a broad-spectrum antiviral,played a crucial role in the treatment of chronic hepatitis C.Recent studies indicate that Rib shows unexpected anti-tumor effects on diverse tumor cells.However,the potential role of Rib via CARM1 and PRMT5 to treat HCC remains largely unexplored.Objective: We will focus on the function of RIB on HCC in vivo and in vitro.To explore whether Rib inhibit HCC growth by regulating CARM1 and PRMT5.We propose that Rib represents a new therapeutic option for HCC patients.Methods:1.Western blot was used to determine the expression of CARM1 and PRMT5 in normal hepatocytes and several HCC cell lines.2.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)and colony assay were used to investigate the effect of Rib in HCC cells in vitro.3.To test the effect of Rib on tumor growth in vivo,we used H22 xenograft mouse model,orthotopic xenograft nude mouse model and H22-induced ascites tumor-bearing model.4.H22-induced ascites tumor-bearing model was used to further evaluate the effect of Rib on peritoneal permeability,the level of VEGF in ascites and the function of Rib on the immune system.5.The effects of Rib on the expression of CARM1,PRMT5 and e IF4 E was analyzed by q RT-PCR and Western blot both in vivo and in vitro.6.We used Ch IP assay to analyze the effects of Rib on the enrichment of CARM1 and H3R17me2a in e IF4 E and VEGF promoter region.Results:1.Western blot results indicated that in comparison with normal hepatocytes,CARM1 and PRMT5 were strongly expressed in HCC cell lines.2.Rib significantly inhibited the proliferation and colony formation in HCC cell lines in vitro.3.Rib not only significantly inhibited tumorigenesis in tumor xenograft mouse model and orthotopic xenograft nude mouse model but also prolong the survival in a H22-induced ascites tumor model in mice effectively.4.Rib reduces peritoneal permeability and VEGF-concentration in ascites.5.Mechanistically,Rib decreased the levels of proliferating cell nuclear antigen(PCNA),CARM1,PRMT5 and their specific histone marks H3R17me2 a,H3R8me2s/H4R3me2 s and e IF4 E.6.Ch IP assay indicated that the enrichment of CARM1 and H3R17me2 a at e IF4 E and VEGF promoter region was decreased by the treatment of Rib.Conclusion: Our results demonstrate that ribavirin not only reduced ascites production but also prolong the survival.Ribavirin inhibited HCC growth both in vitro and in vivo by inhibiting the expression of CARM1 and PRMT5,accumulation of H3R17me2 a and H3R8me2s/H4R3me2 s.In additiona,ribavirin reduced peritoneal permeability and VEGF-concentration in ascites,and enhanced the immune response of the host against the tumor.