Hepatitis B virus X protein mediated suppression of miRNA-122 expression enhances hepatoblastoma cell proliferation through cyclin G1-p53 axis

Abstract

BackgroundHepatitis 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.MethodsExpression 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.ResultsExpression 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.ConclusionOur 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.

Cell cycle checkpoint control: The cyclin G1/Mdm2/p53 axis emerges as a strategic target for broad‑spectrum cancer gene therapy - A review of molecular mechanisms for oncologists

“…By repressing the expression of cyclin G1 ( CCNG1 transcription), miR-122 increases p53 protein levels and suppressor activity and inhibits tumorigenesis in liver cancer models. Insights into the proto-oncogenic function of cyclin G1 were gleaned from studies of HBV subversion in HCC ( 110 ), where it was determined that the HBx-protein directly mediates suppression of miR-122 expression and enhances hepatoblastoma cell proliferation through the functional modulation of the cyclin G1/Mdm2/p53 axis; i.e., downregulation of the expression of the tumor suppressive miR-122 increases cyclin G1 expression, which in turn abolishes p53-mediated suppression of HBV replication and promotes hepatocellular proliferation ( Fig. 9 ; MOA III viral subversion).…”

Host transcription factor Speckled 110 kDa (Sp110), a nuclear body protein, is hijacked by hepatitis B virus protein X for viral persistence

“…Addgene. The 1.3-mer HBV plasmid and 1.3-mer HBV X-null plasmid as used in previous studies (59,60) were obtained as kind gifts. Construct for luciferase assay was done by cloning the promoter region of the STAT1 gene harboring an ISRE site (61) along with the Sp110-binding site in the luciferase expression vector pGL3-Basic.…”

Exploiting Oncogenic Drivers along the CCNG1 Pathway for Cancer Therapy and Gene Therapy

“…Apparently, the major species (∼70% of the total population) of regulatory microRNAs that are commonly lost in the pathogenesis and stage-wise progression of hepatocellular carcinoma is miR-122, which physically “targets” the CCNG1 (cyclin G1) gene for suppression and thus appears to be a natural growth suppressive-microRNA focused on limiting the expression of cyclin G1 in the quiescent stem cells of this potentially proliferative, highly regenerative organ 5 . Turning to virology, a renewed appreciation of the oncogenic potential of dysregulated CCNG1 gene expression—in terms of both persistent stem cell activation ( cell competence ) and overriding p53-mediated checkpoint control (thus driving cell survival over DNA fidelity )—came to light when it was discovered that the carcinogenic hepatitis B virus (HBV) produces a protein, the HBx-protein, that specifically, directly, perhaps s trategically , downregulates the normal expression of miR-122, 6 which results in increased CCNG1 gene expression; raising cyclin G1 to sufficient levels that cyclin-G/PP2A complexes activate Mdm2 and ultimately override the executor-suppressor functions of wild-type p53, thereby abolishing the well-known p53-mediated inhibition of HBV replication as well 6 …”