HBx Protein-mediated ATOH1 Downregulation Suppresses ARID2 Expression And Promotes Hepatocellular Carcinoma

Objective: Hepatitis B virus(HBV)infection is the predominant risk factor for HCC in our country and Hepatitis B virus X protein plays a critical role in the pathogenesis of HBV-related hepatocellular carcinoma.As a multi-functional regulation protein,HBx modulates cellular processes,such as apoptosis,cell cycle progression,cell proliferation,and migration.HBx is able to transactivate and regulate host gene expression by interacting with transcriptional factors.Several studies have reported that a large variety of cellular genes,promoters/enhancers,and transcriptional factors,such as activator protein(AP)-1,nuclear factor kappaB(NF-?B),cAMP response element binding protein(CREB),and activating transcription factor(ATF)/cAMP,are involved in HBx-induced hepatocarcinogenesis.However,the underlying mechanisms through which HBx regulates gene transcription to induce hepatocarcinogenesis remain largely unknown.AT-rich DNA interaction(ARID2),a subnit of PBAF(Polybromo-associated BRG1-associated factor),belongs to the switch/sucrosenonfermenting(SWI/SNF)chromatin-remodeling complex.ARID2 contains a conservative N-terminal ARID region and two conservative C-terminal C2H2 Zn-finger motifs,which can bind directly to DNA or interact with proteins and RNA.ARID-containing proteins are involved in cell lineage gene regulation,embryonic development,cell cycle control.ARID2 is also involved in tumor progression.Recently,ARID2 was found to be frequently mutated in various solid tumors,such as HCC,melanoma,nonsmall lung carcinoma,and gastric adenomas.Our previous study has demonstrated that the expression of ARID2 was significantly downregulated in HCC tissues and ARID2 downregulation was involved with hepatoma cell proliferation and migration.In this study,we aim to investigate the regulation of HBx on ARID2 and the molecular mechanism of HBx-induced ARID2 regulation,to further understand the molecular mechanism in the development of HBV-related hepatocellular carcinoma.Methods: 1)In HBV-expressing hepatoma cell lines,HBx-transient and stable-transduced hepatoma cells and HBV transgenic mice,we used western blot assay to detect the expression of ARID2;In HBV-related HCC tissues,IHC were used to observe the ARID2 expression levels and Real-time PCR to detect the relationship between HBx and ARID2;2)In hepatoma cells,we constructed the human ARID2 promoter reporter pGL3-ARID2 and serial 5' end deletion mutations of ARID2 to observe theeffect of HBx on ARID2 promoter luciferase activities and find out the ARID2 promoter region within which HBx mainly targeted,chromatin immunoprecipitation(Ch IP)assays were applied to observe the recruitments of HBx to ARID2 promoter;3)We predicted the transcription factor binding sites in the-1040~-601 nt promoter region of ARID2 gene by using the JASPAR database(http://jaspar.genereg.net/).We then identified the transcription factor binding sites by Dual-luciferase assay,(ChIP)and immuno precipitation(IP)assays;4)Subsequently,in HBx stabletransduced hepatoma cell lines,we observed the influence of ARID2 overexpression on cell proliferation and migration induced by HBx.Results: Western blot assay showed that ARID2 expression levels were significantly decreased in HBV-replicative hepatoma cells and HBV transgenic mice.Among HBV viral-encoded protein,HBx protein markedly downregulatedARID2 expression,whereas HBc protein and HBs protein had little effects on modulation of ARID2 expression.Meanwhile,when compared with wild-type HBV-transfected cells,cells transduced with HBV replicative plasmid containing stop mutation of HBx(X-Stop)has largely enhanced ARID2 expression,furthermore,X-Stop induced ARID2 restoration was partially abolished by overexpression of HBx.HBx-induced inhibition of ARID2 expression was further clarified in both HBx-transient and stable-transduced hepatoma cells.In HBV-related hepatocellular carcinoma tissues,Immunohistochemical staining showedthat positive rate of ARID2 was significantly lower in tumor tissues when compared to paired adjacent non-tumor tissues.Correlation analysis further indicated that the expression levels of HBx were negatively correlated with those of ARID2 in HCC tissues.Dual-luciferase assay showed that the reporter activity of pGL3-ARID2 increased approximately 15-fold when compared with that of the vector plasmid.Ectopic expression of HBx significantly inhibited ARID2 luciferase signaling compared with that of the vector control.Moreover,HBx was found to inhibit ARID2 promoter activity in a concentration-dependent manner.Furthermore,we found that the5'-flanking region located at-1040 to-601 bp upstream of the human ARID2 gene transcription start site may be mainly responsible for transcriptional repression by HBx.ChIP assays showed that HBx could not directly interact with the ARID2 promoter,suggesting that HBx may inhibit ARID2 transcription through an indirect mechanism.By the JASPAR database,Real-Time PCR and Western blot assays,we screened out differential expression transcription factor ATOH1 and found that HBx inhibited ATOH1 expression both at the mRNA and protein level.Dual-luciferase assay showed that the promoter activity of ARID2 mediated by HBx was largely restored by overexpression of ATOH1 relative to the control.Western blot found that ARID2 expression was restored by ATOH1 ectopic expression in HBx-stable hepatoma cells.ChIPassays demonstrated that HBx decreased the recruitment of ATOH1 to the ARID2 promoter.Meanwhile,ATOH1 knockdown inhibited ARID2 expression.Furthermore,HBx failed to inhibit ARID2 promoter activity when the ATOH1 binding site in the ARID2 promoter was mutated,.However,co-IP analysis indicated that anti-HBx antibodies could not immunoprecipitate ATOH1 in hepatoam cells,and vice versa.Thus,HBx may function as a transcriptional repressor of ARID2 expression through inactivation of ATOH1.Transwell and EdU assays demonstrated that ARID2 partially abolished the increased migration and proliferation of hepatoma cell lines mediated by HBx.Meanwhile,Western blot showed that ARID2 significantly enhanced the expression of the epithelial marker E-cadherin in HBx-stable hepatoma cells.Conclusion: HBx downregulatedARID2 through modulation of the transcription factor ATOH1 in hepatoma cells.Moreover,downregulation of ARID2 by HBx contributed to enhanced HCC cell migration and proliferation,thus eventually contributes to HBV-related HCC carcinogenesis.Our findings provide new insights into the molecular mechanisms of HCC progression mediated by HBx.