Hepatitis C Virus NS3 Protein Enhances Hepatocellular Carcinoma Cell Invasion By Promoting PPM1A Ubiquitination And Degradation

Hepatocellular carcinoma(HCC)is one of the most common cancers worldwide,particularly in China.It ranks as the fifth most common malignancy and the second leading cause of cancer deaths worldwide,with more than 695,900 deaths each year.Chronic hepatitis C virus(HCV)infection is one of the most important risk factors for developing HCC.Although some progress has been made,the mechanism underlying HCV-associated hepatocarcinogenesis remains not fully understood.HCV is a positive single-stranded RNA virus with an exclusively cytoplasmic life cycle.Unlike hepatitis B virus(HBV),which is a DNA virus that can induce insertional mutagenesis,HCV does not insert into the host cell genome.Although the inflammation caused by chronic hepatitis C is likely to contribute to the development of HCC,there is strong evidence that one or more of the viral proteins and its involvement in interrupting cellular signaling pathways contribute mostly to tumorigenesis.Protein phosphatase magnesium-dependent 1A(PPM1A),also known as PP2Ca,is a protein phosphatase that belongs to the Protein Phosphatase 2C family.PPM1A has recently emerged as an important tumor suppressor as it can block a range of tumor-centric signaling pathways through protein dephosphorylation.However,the role and regulatory mechanisms of PPM1A in HCV-infected cells have not been reported.In this study,we examined the expression of PPM1A in HCV-infected hepatoma cells and HCV-related HCC tissues,and we determined whether this protein is involved in HCV-related HCC development.We found a direct link between HCV infection and cellular abundance of PPM1A in both hepatoma cells and the HCC tissues.The mechanism by which NS3 downregulates PPM1A abundance was revealed,and the roles of PPM1A in regulating hepatoma cell invasion and migration were assessed in vitro.Together,our findings provide novel evidence on the mechanisms involved in HCV-mediated progression of HCC,which may provide potential candidates for the clinical prevention and treatment of HCV-associated HCC.Our study is composed of the following three parts:Part Ⅰ HCV infection downregulates PPM1A abundancePurpose:we aimed to investigate the expression and subcellular localization of PPM1A in HCV-infected hepatoma cells and HCV-related HCC tissues.Methods:Total,cytoplasmic,and nuclear PPM1A proteins after HCV infection were detected by western blotting.The subcellular localization of PPM1A was determined by immunofluorescence staining.The expression of PPM1A in normal liver and HCV-related HCC tissues was quantified by immunohistochemistry.Results:PPM1A abundance was significantly reduced within 2-3 days of infection with HCV,and the PPM1A level at 5 days after infection was approximately 30%that of uninfected cells.Furthermore,nuclear expression of PPM1A decreased more dramatically than total PPM1A,while the cytoplasmic abundance slightly increased during this period.Immunofluorescence analysis confirmed the strikingly lower abundance of PPM1A in HCV-infected cells.In mock-infected cells,PPM1A was mainly localized in the nucleus.In contrast,in HCV-infected cells,PPM1A showed a striking re-localization to the cytoplasm,in which HCV replicated and completed its life cycle.In addition,compared with the normal liver tissues,significantly lower levels of PPM1A were found in the HCC tumor and adjacent tissues.Conclusion:PPM1A was strongly downregulated and its normal nuclear localization shifted to a mainly cytoplasmic distribution following infection of cultured hepatoma cells with HCV.Significantly lower levels of PPM1A were also found in HCV-related HCC and adjacent tissues than in normal tissuesPart Ⅱ NS3 interacts with PPM1A and promotes ubiquitin-mediated proteasomal degradation of PPM1APurpose:To explore the mechanism underlying the decrease in PPM1A induced by HCV infection or NS3 expression.Methods:It was hypothesized that the reduction in PPM1A abundance might be mediated by one or more viral proteins expressed during infection.Thus,a panel of expression plasmids involving all 10 HCV proteins with N-terminal epitope tags and transfected these into Huh-7 cells to screen for their ability to downregulate PPM1A expression.The effect of NS3 on the abundance and subcellular localization of PPM1A were determined by western blotting and immunofluorescence,respectively.Co-immunoprecipitation experiments were performed to detect the interaction of NS3 with PPM1 A.NS3 deletion constructs plasmid were made to determine the domain(s)of NS3 that mediates its interaction with PPM1A.Finally,the mechanism underlying the decrease in PPM1A induced by HCV infection or NS3 expression was systematically explored.First,PPM1A mRNA levels were measured by qRT-PCR in Huh-7 cells infected with HCVcc for 5 days.Subsequently,we assessed the protein stability of PPM1A in NS3-expressing cells after treatment with cycloheximide,a protein synthesis inhibitor.Treatment of NS3-transfected cells with UPP inhibitor MG132 or ALP inhibitor chloroquine were used to analyze which protein degradation pathway was affected by NS3.Since ubiquitination is generally enhanced prior to proteasomal degradation,we examined whether HCV infection and NS3 expression enhanced the ubiquitination of PPM1A with the employment of precipitation with anti-PPM1A and immunoblotting with either anti-ubiquitin or anti-PPM1A antibody.Results:Transient transfection of NS3,but not other HCV proteins,significantly reduced PPM1A expression.NS3 interacts with endogenous PPM1A in Huh-7 cells and exogenously expressed PPM1A in 293T cells,which is dependent on its protease domain.PPM1A mRNA was upregulated in HCV-infected cells from 2 days after infection.Similarly,ectopic expression of NS3 significantly elevated the PPM1A mRNA level,likely owing to the compensation.In addition,the stability of the PPM1A protein was decreased in NS3 expressed cells after treatment with cycloheximide,a protein synthesis inhibitor.Together,these results indicated that NS3 modulates the level of PPM1A via posttranscriptional regulation.Furthermore,treatment of NS3-transfected cells with UPP inhibitor MG132 or ALP inhibitor chloroquine revealed that MG132 almost completely abolished the effect of NS3 on the PPM1A protein level.This indicated that NS3 might affect the proteasomal degradation of PPM 1 A.Lastly,the levels of ubiquitinated PPM1A were significantly higher in HCV-infected and NS3 overexpressed cells than in its control cells.Conclusion:HCV protein NS3 is responsible for the modulation of the abundance and cellular localization of PPM1A.NS3 interacts with PPM1A through its protease domain and downregulates PPM1A protein level by promoting its ubiquitin-dependent proteasomal degradation.Part Ⅲ PPM1A overexpression reverses NS3-mediated enhancement on cell invasionPurpose:To explore the roles of NS3 and PPM1A in hepatoma cell migration and invasion,and to prove that downregulation of PPM1A is involved in NS3-induced promotion of HCC metastasisMethods:We used RNA interference to examine the impact of PPM1A knockdown on Huh-7 cell migration and invasion using wound healing and transwell invasion assays.In addition,we restored the level of PPM1A in cells expressing NS3 and then measured the cell migration and invasion capacities.Results:NS3 expression or blockade of PPM1A significantly promoted HCC cell migration,invasion,and epithelial mesenchymal transition(EMT),which were further intensified by TGF-β1 stimulation,in vitro.Furthermore,restoration of PPM1A abrogated the NS3-mediated promotion of HCC migration and invasion to a great extent,which was dependent on its protein phosphatase function.Conclusion:NS3 expression or loss of PPM1A significantly promoted HCC cell migration,invasion,and EMT,and the cellular invasion capacity caused by NS3 is mediated at least partially through the modulation of PPM1A abundance.