MOF Upregulates Estrogen Receptor α Signaling Pathway Via Its Acetylase Activity In Hepatocellular Carcinoma

Objective: Primary liver cancers include hepatocellular carcinoma(HCC)(75%-85% of cases)and intrahepatic cholangiocarcinoma(10%-15% of cases)and other rare types.There are approximately 841,000 new cases and 782,000 deaths each year.In most parts of the world,the incidence and mortality of men are two to three times higher than women.The incidence of HCC increases in postmenopausal women,and estrogen therapy can suppress this phenomenon.The prognosis of women with HCC is better than that of men.Currently,only sorafenib and Lenvatinib are approved for firstline treatment of advanced,unresected HCC,but only produce a degree of survival benefit.Therefore,new therapeutic targets need to be identified to improve current HCC treatments.Previous studies have shown that ERα is a member of the nuclear receptor superfamily of steroid hormones.ERα forms a complex with ligands and transcriptional co-regulators and binds to the response elements of target genes,and regulates gene transcription through mechanisms such as changing chromosome structure.This indicates that the study of new co-regulatory factors of ERα or the mechanism of regulating the post-translational modification of ERα will provide experimental evidence and new targets for the early diagnosis and treatment of hepatocellular carcinoma.Previous studies have found that MOF,as an important histone H4K16 acetyltransferase,plays an important tumor suppressor function in the occurrence and development of most primary tumors.In addition to histone acetylation modification,MOF can also acetylate certain non-histone proteins,such as P53,FASN,LSD1,No RC and IRF3.Previous studies have shown that MOF is lower in hepatocellular carcinoma.The prognosis of patients with lower MOF expression is worse.However,the mechanism of how MOF exerts its anti-cancer effect is still unclear.The early research of our research group reported that the combination of MOF and ERα plays a tumor suppressor effect in endometrial cancer.This article aims to study in depth the biological function of MOF in HCC and the molecular mechanism of MOF stabilizing ERα protein and acting as a coregulator of ERα in HCC.Studying the mechanism of MOF in HCC is expected to provide a reliable experimental basis for the early diagnosis and new target treatment of HCC patients.Methods: 1.This study uses bioinformatics methods to predict whether MOF will affect the prognosis of clinical patients with hepatocellular carcinoma;secondly,analyze the m RNA expression level of MOF in hepatocellular carcinoma;Immunohistochemical(IHC)experiments were performed to detect the expression of MOF in 145 cases of hepatocellular carcinoma and 48 cases of adjacent non-cancerous tissues in clinical specimens;Western blot experiments were performed to detect the expression of MOF and ERα in 33 pairs of hepatocellular carcinoma and adjacent tumors.2.Co-immunoprecipitation experiments(Co-IP)and immunofluorescence confocal microscope scanning technology were performed to determine whether there is an interaction between MOF and ERα;q PCR and western blot experiments were performed to detect the effect of MOF overexpression or knockdown on ERα m RNA and protein.3.The acetylation modification experiment method was performed to detect the effect of MOF overexpression or knockdown on the acetylation level of ERα;ubiquitination modification experiment method was performed to detect the effect of MOF overexpression or knockdown or MOF mutant plasmid on ERα ubiquitination level.4.Dual luciferase reporter experiments were performed to explore the regulation of MOF overexpression or knockdown on ERα mediated transactivation;Real-time PCR and Western blot experiments were performed to verify whether ERα downstream target genes are regulated by MOF;Chromatin immunoprecipitation experiments(Ch IP)were performed to verify whether ERα or MOF are recruited to the response element ERE of ERα target genes.5.In order to further study the role of MOF in hepatocellular carcinoma,clone formation,cell proliferation,migration and invasion experiments were performed to explore the biological functions of MOF on hepatocellular carcinoma cell lines.6.In order to further study the effect of MOF on hepatocellular carcinoma in vivo,the mouse subcutaneous tumor formation experiment was performed to verify the effect of MOF on hepatocellular carcinoma proliferation.Results: 1.This study confirms that the clinical hepatocellular carcinoma patients with higher MOF expression have the better prognosis;secondly,the m RNA expression level of MOF in hepatocellular carcinoma was lower than that in non-cancerous tissues;higher expression of MOF was significantly associated with well differentiation;the expression of MOF and ERα in fresh liver cancer tissue samples was decreased and there was a positive correlation.2.MOF and ERα interacted in hepatocellular carcinoma cell lines;MOF and ERα co-localized in the cell,mainly co-localized in the nucleus;MOF overexpression stabilized ERα protein.3.MOF acetylated ERα at K266,K268,and K299;MOF acetyltransferase inhibits agent MG149 destabilizes ERαprotein;MOF overexpression or knockdown can inhibit or promote the level of ERαubiquitination.4.MOF up-regulates ERα-mediated transactivation;MOF up-regulates ERα downstream endogenous target gene(SHP and SMAD7)regulation;ERα or MOF is recruited to the response element EREIII of the ERα target gene SMAD7.5.MOF inhibited the growth,migration,invasion and lipid droplet formation of hepatocellular carcinoma cell lines.6.MOF inhibited the proliferation of hepatocellular carcinoma in vivo.Conclusion: 1.MOF is lower in HCC tissues and has a positive correlation with ERα.2.MOF stabilizes ERα protein but does not affect the m RNA level of ERα.3.MOF inhibits the polyubiquitination level of ERα by acetylating ERα at K266,K268,K299.4.MOF up-regulates ERα-mediated transactivation and up-regulates the m RNA and protein levels of endogenous ERα target genes.5.MOF inhibits cell growth of hepatocellular carcinoma,partly dependent on ERα;MOF inhibits cell migration and invasion of hepatocellular carcinoma.6.MOF inhibits cell growth of hepatocellular carcinoma in vivo.