title: Hepatitis B virus X protein downregulates expression of the miR-16 family in malignant hepatocytes in vitro.
abstract: BACKGROUND
  Hepatitis B virus X protein (HBx) is involved in the initiation and progression of hepatocellular carcinoma (HCC) by regulating the host protein-coding genes. In this study, we showed that HBx altered the expression of microRNAs (miRNAs) to promote proliferation and transformation in malignant hepatocytes in vitro.
  
  
  METHODS
  miRNA microarray and quantitative reverse-transcription polymerase chain reactions (qRT-PCRs) were performed to identify miRNAs that were differentially regulated by HBx in HCC cells. Protein, mRNA, and miRNA expression analyses; cell cycle and apoptosis analyses; loss/gain-of-function analysis; and luciferase reporter assays were performed to delineate the consequences of miR-16 family repression in HepG2 cells.
  
  
  RESULTS
  Hepatitis B virus X protein induced widespread deregulation of miRNAs in HepG2 cells, and the downregulation of the miR-16 family was reproducible in HepG2, SK-HEP-1, and Huh7 cells. CCND1, a target of the miR-16 family, was derepressed by HBx in HepG2 cells. c-Myc mediated the HBx-induced repression of miR-15a/16 in HepG2 cells. Ectopically expressed miR-15a/16 suppressed the proliferation, clonogenicity, and anchorage-independent growth of HBx-expressing HepG2 cells by arresting them in the G1 phase and inducing apoptosis, whereas reduced expression of miR-16 accelerated the growth and cell-cycle progression of HepG2 cells.
  
  
  CONCLUSIONS
  Hepatitis B virus X protein altered the in vitro expression of miRNAs in host malignant hepatocytes, particularly downregulating the miR-16 family. Repression of miR-15a/16 is c-Myc mediated and is required for the HBx-induced transformation of HepG2 cells in vitro. Therefore, miR-16 family may serve as a therapeutic target for hepatitis B virus (HBV)-associated HCC.
authors.affiliation: Department of Hepatobiliary Surgery, Sun-Yat-Sen Memorial Hospital, Sun-Yat-Sen University, 107 Yanjiang West Road, Guangzhou 510120, People's Republic of China.
authors.forename: G
authors.initials: G
authors.lastname: Wu
authors.forename: F
authors.initials: F
authors.lastname: Yu
authors.forename: Z
authors.initials: Z
authors.lastname: Xiao
authors.forename: K
authors.initials: K
authors.lastname: Xu
authors.forename: J
authors.initials: J
authors.lastname: Xu
authors.forename: W
authors.initials: W
authors.lastname: Tang
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: E
authors.initials: E
authors.lastname: Song
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D049109:Cell Proliferation; D002478:Cells, Cultured; D015536:Down-Regulation; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
chemical_list: C505362:MIRN16 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D008156:Luciferases