title: Hepatitis B virus X protein mediated suppression of miRNA-122 expression enhances hepatoblastoma cell proliferation through cyclin G1-p53 axis.
abstract: BACKGROUND
  Hepatitis B virus (HBV) X protein (HBx) reported to be associated with pathogenesis of hepatocellular carcinoma (HCC) and miR-122 expression is down regulated in HCC. Previous studies reported miR-122 targets cyclin G1 (CCNG1) expression and this in turn abolishes p53-mediated inhibition of HBV replication. Here we investigated the involvement of HBx protein in the modulation of miR-122 expression in hepatoblastoma cells.
  
  
  METHODS
  Expression of miR-122 was measured in HepG2 cells transfected with HBx plasmid (HBx-HepG2), full length HBV genome (HBV-HepG2) and in constitutively HBV synthesizing HepG2.2.15 cells. CCNG1 mRNA (a direct target of miR-122) and protein expressions were also measured in both HBx-HepG2, HBV-HepG2 cells and in HepG2.2.15 cells. miR-122 expressions were analyzed in HBx-HepG2, HBV-HepG2 and in HepG2.2.15 cells after treatment with HBx mRNA specific siRNA. Expressions of p53 mRNA and protein which is negatively regulated by CCNG1 were analyzed in HBx transfected HepG2 cells; X silenced HBx-HepG2 cells and X silenced HepG2.2.15 cells. HBx induced cell proliferation in HepG2 cells was measured by cell proliferation assay. Flow cytometry was used to evaluate changes in cell cycle distribution. Expression of cell cycle markers were measured by real time PCR.
  
  
  RESULTS
  Expression of miR-122 was down regulated in HBx-HepG2, HBV-HepG2 and also in HepG2.2.15 cell line compared to control HepG2 cells. CCNG1 expression was found to be up regulated in HBx-HepG2, HBV-HepG2 cells and in HepG2.2.15 cells. Following siRNA mediated silencing of HBx expression; increased miR-122 levels were documented in HBx-HepG2, HBV-HepG2 and in HepG2.2.15 cells. HBx silencing in HBx-HepG2 and HepG2.2.15 cells also resulted in increased p53 expression. FACS analysis and assessment of expressions of cell cycle markers revealed HBx induced a release from G1/S arrest in HepG2 cells. Further, cell proliferation assay showed HBx promoted proliferation of HepG2 cell.
  
  
  CONCLUSIONS
  Our study revealed that HBx induced down regulation of miR-122 expression that consequently increased CCNG1 expression. This subsequently caused cell proliferation and release from G1/S arrest in malignant hepatocytes. The study provides the potential to utilize the HBx-miR-122 interaction as a therapeutic target to limit the development of HBV related HCC.
authors.affiliation: ICMR Virus Unit, Kolkata, Indian Council of Medical Research, GB-4, 1st floor, ID & BG Hospital Campus, 57, Dr. S C Banerjee Road, Beliaghata, Kolkata, 700010 West Bengal India.
authors.forename: Manikankana
authors.initials: M
authors.lastname: Bandopadhyay
authors.affiliation: ICMR Virus Unit, Kolkata, Indian Council of Medical Research, GB-4, 1st floor, ID & BG Hospital Campus, 57, Dr. S C Banerjee Road, Beliaghata, Kolkata, 700010 West Bengal India.
authors.forename: Neelakshi
authors.initials: N
authors.lastname: Sarkar
authors.affiliation: Molecular Virology Laboratory, Defense Research Laboratory (DRDO), Tezpur, Assam India.
authors.forename: Sibnarayan
authors.initials: S
authors.lastname: Datta
authors.affiliation: ICMR Virus Unit, Kolkata, Indian Council of Medical Research, GB-4, 1st floor, ID & BG Hospital Campus, 57, Dr. S C Banerjee Road, Beliaghata, Kolkata, 700010 West Bengal India.
authors.forename: Dipanwita
authors.initials: D
authors.lastname: Das
authors.affiliation: ICMR Virus Unit, Kolkata, Indian Council of Medical Research, GB-4, 1st floor, ID & BG Hospital Campus, 57, Dr. S C Banerjee Road, Beliaghata, Kolkata, 700010 West Bengal India.
authors.forename: Ananya
authors.initials: A
authors.lastname: Pal
authors.affiliation: ICMR Virus Unit, Kolkata, Indian Council of Medical Research, GB-4, 1st floor, ID & BG Hospital Campus, 57, Dr. S C Banerjee Road, Beliaghata, Kolkata, 700010 West Bengal India ; Present Address: Department of Pathology & Lab Medicine, Tulane University School of Medicine, New Orleans, LA 70112 USA.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Panigrahi
authors.affiliation: ICMR Virus Unit, Kolkata, Indian Council of Medical Research, GB-4, 1st floor, ID & BG Hospital Campus, 57, Dr. S C Banerjee Road, Beliaghata, Kolkata, 700010 West Bengal India.
authors.forename: Arup
authors.initials: A
authors.lastname: Banerjee
authors.affiliation: Chittaranjan National Cancer Institute, 37, SP Mukherjee Road, Kolkata, India.
authors.forename: Chinmay K
authors.initials: CK
authors.lastname: Panda
authors.affiliation: Saha Institute of Nuclear Physics, Bidhan nagar, Kolkata India.
authors.forename: Chandrima
authors.initials: C
authors.lastname: Das
authors.affiliation: National Institute of Cholera and Enteric Diseases, Kolkata, India.
authors.forename: Shekhar
authors.initials: S
authors.lastname: Chakrabarti
authors.affiliation: ICMR Virus Unit, Kolkata, Indian Council of Medical Research, GB-4, 1st floor, ID & BG Hospital Campus, 57, Dr. S C Banerjee Road, Beliaghata, Kolkata, 700010 West Bengal India.
authors.forename: Runu
authors.initials: R
authors.lastname: Chakravarty
keywords: CCNG1; HBx; HepG2; HepG2.2.15; Hepatocellular carcinoma; miR-122