Aberrant Expression Of C19MC Cluster And Mir-1269b Induced By Hepatitis B Virus X Protein In Hepatoma Cells

[Objective]HepatocelIular carcinoma is the most common primary liver cancer in the malignant tumor and the third most common cause of cancer-related death worldwide after gastric cancer,esophageal cancer.HCC is closely related to HBV infection.MicroRNAs(miRNAs)are 21-23nt endogenous regulatory noncoding RNAs in all multicellular eukaryotes and strongly associated with various diseases,including cancer.Previous studies have indicated HBV can modulate the expression of cellular miRNAs in HBV-related hepatocellular carcinoma.However,the mechanism underlying HBV regulating miRNAs is not clear yet.In this study,we found C19MC and miR-1269b differentially expressed in HBV negative and HBV positive HCC tissue through deep sequencing.The purpose of this study is to clarify the mechanisms underlying aberrant expression of C19MC cluster and miR-1269b in Hepatoma cells.[Methods]First,we found differentially expressed miRNAs in HBV negative and HBV positive HCC tissue through deep sequencing.We selected the miRNAs,C19MC and miR-1269b.Using the qRT-PCR method,we investigated aberrant expression of C19MC and miR-1269b in HCC tissue and Hepatoma cells.And HBx regulated the expression of C19MC and miR-1269b.Next,bioinformatics analysis was performed to pridict CpG island in the upstream of C19MC.Then,we assessed the expression of C19MC in 5-aza-2’-deoxycytidine(5-Aza-CdR)treated HepG2.2.15 and whether HBx could increase the expression of DNMT3A and DNMT1 by qRT-PCR.In addition,we detected pri-miR-1269b levels in HepG2 cells transfected with HBV 1.3 and HBx.And we predicted and constucted the promoter of miR-1269b.Using dual luciferase report system,we verified whether the promoter was work in 293T cells and HepG2 cells and the effect of HBV 1.3 and HBx on miR-1269b gene promoter luciferase reporter activity.As miR-1269b gene promoter contains no CpG island,we predicted NF-kappa B and p53 localizes within the mir-1269b gene transcriptional element.To further investigate the relationship between miR-1269b and NF-kappa B/p53,we performed dual luciferase report assay to determine miR-1269b gene promoter luciferase reporter activity in cells transfected with NF-kappa B or p53.To further determine NF-kappa B bind to the miR-1269b promoter,we generated a luciferase reporter construct without NF-kappa B response element of the miR-1269b promoter.And qRT-PCR was used to detect the effect of NF-kappa B on miR-1269b expression.[Results]qRT-PCR results showed that the expression of C19MC and miR-1269b was down-regulated by HBV.What is more,HepG2 and Huh7 were transfected with HBx plasmid,followed by qRT-PCR and C19MC and miR-1269b were found to be down-regulated.These results suggested that HBV represses C19MC and miR-1269b expression through HBx.Next,bioinformatics analysis and qRT-PCR results revealed that C19MC is regulated by the CpG island in the upstream of C19MC.And transfection with HBx in HepG2 cells could increase the expression of DNMT3A and DNMT1.Using qRT-PCR,we found pri-miR-1269b levels decreased in HepG2 cells transfected with HBV 1.3 and HBx.Then,we predicted and successfully constucted the promoter of miR-1269b.Using dual luciferase report system,we verified the promoter was work in 293T cells and HepG2 cells.And HBV infection and HBx down-regulated miR-1269b gene promoter transcription activity.As miR-1269b gene promoter contains no CpG island,we further demonstrated that NF-kappa B down-regulates miR-1269b gene promoter transcription activity.To further determine NF-kappa B bind to the miR-1269b promoter,we generated a luciferase reporter construct without NF-kappa B response element of the miR-1269b promoter.And,we verified NF-kappa B binds to the miR-1269b promoter directly.In addition,we found NF-kappa B can suppress miR-1269b expression.[Conclusions]Our findings provide novel evidence for HBx mediated inhibition of C19MC and miR-1269b transcription by increasing DNMT and NF-kappa B.In addition,this is the first report HBx down-regulates the expression of miR-1269b.Our data also provides important mechanistic insight into differential regulation of micorRNAs by HBV and the relationship between HBV and HCC development.