Regulatory Mechanisms And Biological Significance Of HCV NS5A-mediated Down-regulation Of GADD45α And PTEN Expression

Background and Aim:The hepatitis C virus (HCV) non-structural5A (NS5A) protein plays an important role in cell survival and is linked to the development of hepatocellular carcinoma (HCC). However, the role of HCV NS5A in the development of HCC remains to be clarified. Growth arrest and DNA damage45-alpha (GADD45a) has been shown to be a tumor suppressor and is implicated in cell cycle arrest and suppression of cell growth. Phosphatase and tensin homolog deleted in chromosome10(PTEN) also belongs to tumor suppressor and is involved in cellular processes, including tumorigenesis and cell apoptosis in the liver. Clininc studies have reported that decreased levels of GADD45a and PTEN are associated with HCC pathogenesis and poor prognosis in HCV-infected HCC patients. However, the molecular processs governing the reduction of GADD45a and PTEN as well as outcome of GADD45a and PTEN dysfunction in hepatocytes are unknown. We sought to observe whether GADD45a mediates HCV NS5A-induced cellular survival and to elucidate the molecular mechanism of GADD45a expression regulated by HCV NS5A. We also assessed PTEN expression in the cells harboring with HCV replication and HCV-infected cells. Furthermore, we explored the mechanism of this phenotype and outcomes of PTEN dysfunction in human hepatoma cells.Methods:The GADD45a and PTEN promoter activity, mRNA transcript levels, and protein levels were assessed using luciferase assays, real-time PCR, and western blotting, respectively. Ectopic expression of p53and small interfering RNAs (siRNAs) for targeting p53, NF-κB, and PI3K were used to explore the mechanism by which HCVNS5A regulates GADD45α expression. In addition, cell viability and colony formation assays were performed to evaluate cell proliferation in human hepatoma cells in the presence of GADD45a knockdown by siRNAs or ectopic expression of GADD45a. Using pharmacological inhibitors and siRNAs, we explored the mechanism by which HCVNS5A regulated PTEN expression. Moreover, Caspase3/7activity assays were performed as well as Caspase-3and PARP protein were assessed to evaluate cells apoptosis in the presence of PTEN over-expression or Akt inhibition in human hepatoma cells.Results:We found that HCV NS5A down-regulated GADD45a expression at the transcriptional level by decreasing the promoter activity, mRNA transcription, and protein levels. Knockdown of p53resulted in a similar decrease in GADD45a expression as that caused by HCV NS5A. Overexpression of p53reversed the HCV NS5A-mediated down-regulation of GADD45a. HCV NS5A repressed p53expression, which was followed by a subsequent decrease of GADD45a expression. We further provide evidence that HCV NS5A led to increases of phosphor-NF-κB and Akt levels. Inhibition of these pathways using pharmacological inhibitors or specific siRNAs rescued HCV NS5A-mediated down-regulation of p53and GADD45a. We also found that HCV NS5A protein and depletion of GADD45a increased cell growth, whereas ectopic expression of GADD45a abrogated HCV NS5A-induced cell proliferation. Additionally, we found that HCV down-regulated PTEN promoter activity, mRNA levels, and protein expression in cultured human hepatoma cells. We further identified NS5A protien as a key determinant for PTEN reduction among HCV proteins. HCV NS5A led to increases of phosphor-NF-κB and Akt levels through ROS-dependent and-independent manners, respectively. Inhibition of these pathways using pharmacological inhibitors or specific siRNAs for targeting NF-κB and PI3K rescued HCV NS5A-mediated down-regulation of PTEN. Moreover, PTEN and PI3K were mutually antagonistic to Akt activation. PTEN reduction enhanced PI3K-induced Akt activation. We also found that HCV NS5A suppressed cellular apoptosis, whereas ectopic expression of PTEN or inhibition of Akt abrogated HCV NS5A-mediated inhibition of cellular apoptosis.Conclusions:The current study shows that HCV NS5A down-regulates GADD45a expression and subsequently triggers cellular proliferation. HCV NS5A down-regulates PTEN expression through ROS-dependent and-independent manners, consequently contributing to inhibition of cell apoptosis. These findings provide new insights suggesting that HCV NS5A could contribute to HCV-related HCC occurrence.