Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar 10;36(1):42.
doi: 10.1186/s13046-017-0510-8.

Hepatitis C virus NS3 protein enhances hepatocellular carcinoma cell invasion by promoting PPM1A ubiquitination and degradation

Yali Zhou  1 , Yan Zhao  1 , Yaoying Gao  1 , Wenjun Hu  1 , Yan Qu  1 , Ning Lou  2 , Ying Zhu  3 , Xiaoping Zhang  4 , Hongmei Yang  5
Affiliations

Hepatitis C virus NS3 protein enhances hepatocellular carcinoma cell invasion by promoting PPM1A ubiquitination and degradation

Yali Zhou et al. J Exp Clin Cancer Res. .

Abstract

Background: Growing evidence suggests that hepatitis C virus (HCV) contributes to hepatocellular carcinoma (HCC) by directly modulating oncogenic signaling pathways. Protein phosphatase magnesium-dependent 1A (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.

Methods: Total, cytoplasmic, and nuclear PPM1A protein after HCV infection or overexpression of HCV nonstructural protein 3 (NS3) were detected by western blotting. The expression of PPM1A in normal liver and HCV-related HCC tissues was quantified by immunohistochemistry. The effects of HCV infection and NS3 expression on the PPM1A protein level were systematically analyzed, and the ubiquitination level of PPM1A was determined by precipitation with anti-PPM1A and immunoblotting with either anti-ubiquitin or anti-PPM1A antibody. Finally, the roles of NS3 and PPM1A in hepatoma cell migration and invasion were assessed by wound healing and transwell assays, respectively.

Results: HCV infection and replication decreased PPM1A abundance, mediated by NS3, in hepatoma cells. Compared to normal liver tissues, the expression of PPM1A was significantly decreased in the HCC tumor tissues and adjacent non-tumor tissues. NS3 directly interacted with PPM1A to promote PPM1A ubiquitination and degradation, which was dependent on its protease domain. Blockade of PPM1A through small interfering RNA 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.

Conclusions: Our findings demonstrate that the HCV protein NS3 can downregulate PPM1A by promoting its ubiquitination and proteasomal degradation, which might contribute to the migration and invasion of hepatoma cells and may represent a new strategy of HCV in carcinogenesis.

Keywords: Cancer cell invasion; Hepatitis C virus; Hepatocellular carcinoma; PPM1A; Ubiquitination and degradation.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
PPM1A abundance is negatively regulated in HCV-infected cells. a Schematic representation of the experiments shown in (b–e). Huh-7 cells were infected with JFH1 virus for 0–5 days at 1 multiplicity of infection (MOI) at intervals, and subsequently lysed simultaneously for immunoblot analysis. Hollow bars show uninfected cells; shaded bars, infected cells. b Levels of HCV infection in Huh-7 cells were determined by immunoblotting for HCV NS5A and core protein as described in a. c–e Total (c), nuclear (d), and cytoplasmic (e) PPM1A protein levels were measured by western blotting. The bar graph (mean ± SEM) displays protein quantification (n = 3). Data are normalized to a loading control (tubulin for total and cytoplasmic PPM1A, and lamin A/C for nuclear PPM1A) and expressed as the fold change relative to the protein levels in uninfected cells. f Cells were infected or not with JFH1 for 3 days, and the subcellular localization of PPM1A was monitored by dual immunolabeling of HCV core protein (green) and PPM1A (red). Arrows denote HCV-infected cells in which PPM1A expression and subcellular localization are significantly changed. g Immunohistochemistry was used to measure the expression of PPM1A (brown staining) in normal liver tissues (n = 10), and HCV-related HCC tissues and adjacent tissues (n = 12 each). g Immunohistochemistry was used to measure the expression of PPM1A (brown staining) in normal liver tissues (n = 10), and HCV-related HCC tissues and adjacent tissues (n = 12 each). Arrows indicate representative staining of PPM1A. Left panels show representative images of PPM1A expression, quantitative data are shown in the right panel. Data are the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 as evaluated using Student’s t-test or one-way ANOVA
Fig. 2
Fig. 2
NS3 is responsible for the regulation of PPM1A abundance. a–c Huh-7 cells were transiently transfected with plasmid encoding one of the indicated HCV proteins (a), Flag-vector or Flag-NS3 plasmids at different concentrations as indicated (b), and NS3 or NS3 S139A plasmids (c) for 48 h and subsequently collected for western blotting. d Huh-7 cells were transfected with GFP-vector, GFP-NS3 or GFP-protease for 48 h and subsequently stained for PPM1A (red); the intracellular localization of PPM1A was examined by fluorescence microscopy
Fig. 3
Fig. 3
NS3 protein interacts with PPM1A. a–c Huh-7 cells were transiently transfected with Flag-NS3 or vector (a); HEK293T cells were cotransfected with Flag-PPM1A and GFP-NS3 (b). HEK293T cells were cotransfected with Flag-PPM1A and GFP-protease or GFP-helicase (c), 36 h later, co-IP was performed to identify the interaction between PPM1A and NS3 or its deletion mutants
Fig. 4
Fig. 4
HCV infection and NS3 expression promote the degradation of PPM1A via the ubiquitin proteasome pathway. a Huh-7 cells were infected with JFH1 for 0–5 days as described in Fig. 1a (left panel). The cells were transfected with vector or NS3 for 36 h (right panel), and subsequently, the PPM1A mRNA level was determined by qRT-PCR. Data (n = 3) were normalized to GAPDH and presented as the fold change as compared with control cells. b–c Huh-7 cells were transfected with vector or NS3 for 36 h and subsequently treated with CHX (300 μM) for 0–3 h as indicated (b), vehicle (DMSO), MG132 (20 μM) or chloroquine (50 μM) for 12 h (c). PPM1A protein levels were detected by immunoblotting. d–e Huh-7 cells were infected with JFH1 virus (MOI ~0.5) for 72 h (d), transfected with Flag-vector or Flag-NS3 for 36 h (e), and treated with MG132 (20 μM) for 12 h. The cells were lysed and subjected to immunoprecipitation with antibody against PPM1A and analyzed by western blotting with antibodies against the indicated proteins. Arrows in the anti-ubquitin blots indicate the position of unmodified PPM1A proteins
Fig. 5
Fig. 5
Restoration of PPM1A abundance partially reverses NS3-mediated enhancement of cell invasion. a–b After transfection with vector, NS3, NS3 and PPM1A, NS3 and PPM1A D239N, respectively, the migration and invasion capacities of Huh-7 were analyzed by wound closure (a) and transwell invasion (b) assays. c The protein levels of PPM1A, NS3, vimentin, E-cadherin and N-cadherin were measured by western blotting after transfection with plasmids indicated above. Data are the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 as evaluated using Student’s t-test
Fig. 6
Fig. 6
Knockdown of PPM1A promotes the invasive capacity of HCC cells, which is intensified by exogenous TGF-β1. a–b mRNA (a) and protein (b) levels of PPM1A in Huh-7 cells were assessed by qRT-PCR and western blotting, respectively. Huh-7 cells were transfected with 100 nM PPM1A siRNA (si-PPM1A) or control siRNA (si-NC) for 36 h before detection. GAPDH and tubulin served as internal controls for mRNA and protein loading, respectively. c–d Huh-7 cells were transfected with si-PPM1A or si-NC and treated with TGF-β1 (200 pM) or vehicle. Wound healing (c) and transwell invasion (d) assays were performed to analyze the migration and invasion ability of Huh-7 cells. e Western blot analysis for vimentin, E-cadherin, N-cadherin protein in Huh-7 cells following transfection with si-PPM1A or si-NC. Data are the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 as evaluated using Student’s t-test
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bosch FX, Ribes J, Diaz M, Cleries R. Primary liver cancer: worldwide incidence and trends. Gastroenterology. 2004;127(5 Suppl 1):S5–s16. doi: 10.1053/j.gastro.2004.09.011. - DOI - PubMed
    1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90. doi: 10.3322/caac.20107. - DOI - PubMed
    1. Lavanchy D. The global burden of hepatitis C. Liver Int. 2009;29(Suppl 1):74–81. doi: 10.1111/j.1478-3231.2008.01934.x. - DOI - PubMed
    1. Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat Rev Microbiol. 2007;5(6):453–63. doi: 10.1038/nrmicro1645. - DOI - PubMed
    1. Jeong SW, Jang JY, Chung RT. Hepatitis C virus and hepatocarcinogenesis. Clin Mol Hepatol. 2012;18(4):347–56. doi: 10.3350/cmh.2012.18.4.347. - DOI - PMC - PubMed

Publication types

Substances