Loss of nuclear PTEN in HCV-infected human hepatocytes

Background Hepatitis C virus (HCV) infection is a major risk factor for chronic hepatitis and hepatocellular carcinoma (HCC); however, the mechanism of HCV-mediated hepatocarcinogenesis is not well understood. Insufficiency of PTEN tumor suppressor is associated with more aggressive cancers, including HCC. We asked whether viral non-coding RNA could initiate oncogenesis in HCV infected human hepatocytes. The results presented herein suggest that loss of nuclear PTEN in HCV-infected human hepatocytes results from depletion of Transportin-2, which is a direct target of viral non-coding RNA, vmr11. Methods The intracellular distribution of PTEN in HCV-infected cells was monitored by immunostaining and Western blots of nuclear and cytoplasmic proteins. Effects of PTEN depletion were examined by comparing expression arrays of uninfected cells with either HCV-infected or vmr11-transfected cells. Target genes suggested by array analyses were validated by Western blot. The influence of nuclear PTEN deficiency on virus production was determined by quantitative analysis of HCV genomic RNA in culture media of infected hepatocytes. Results Import of PTEN to the nucleus relies on the interaction of Transportin-2 and PTEN proteins; we show that depletion of Transportin-2 by HCV infection or by the introduction of vmr11 in uninfected cells results in reduced nuclear PTEN. In turn, nuclear PTEN insufficiency correlates with increased virus production and the induction of ?-H2AX, a marker of DNA double-strand breaks and genomic instability. Conclusion An HCV-derived small non-coding RNA inhibits Transportin-2 and PTEN translocation to the nucleus, suggesting a direct viral role in hepatic oncogenesis

Role of hepatitis C virus inducted osteopontin in epithelial to mesenchymal transition, migration, and invasion of hepatocytes

Osteopontin (OPN) is a secreted phosphoprotein which has been linked to tumor progression and metastasis in a variety of cancers including hepatocellular carcinoma (HCC). Previous studies have shown that OPN is upregulated during liver injury and inflammation. However, the role of OPN in hepatitis C virus (HCV)-induced liver disease pathogenesis is not known. In this study, we determined the induction of OPN, and then investigated the effect of secreted forms of OPN in epithelial to mesenchymal transition (EMT), migration and invasion of hepatocytes. We show the induction of OPN mRNA and protein expression by HCV-infection. Our results also demonstrate the processing of precursor OPN (75 kDa) into 55 kDa, 42 kDa and 36 kDa forms of OPN in HCV-infected cells. Furthermore, we show the binding of secreted OPN to integrin αVβ3 and CD44 at the cell surface, leading to the activation of downstream cellular kinases such as focal adhesion kinase (FAK), Src, and Akt. Importantly, our results show the reduced expression of epithelial marker (E-cadherin) and induction of mesenchymal marker (N-cadherin) in HCV-infected cells. We also show the migration and invasion of HCV-infected cells using wound healing assay and matrigel coated Boyden chamber. In addition, we demonstrate the activation of above EMT markers, and the critical players involved in OPN-mediated cell signaling cascade using primary human hepatocytes infected with Japanese fulminant hepatitis (JFH)-1 HCV. Taken together, these studies suggest a potential role of OPN in inducing chronic liver disease and HCC associated with chronic HCV infection

HCV promotes invasion of hepatoma cells.

<p>(A) HCV-infected cells (from the same pool of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087464#pone-0087464-g002" target="_blank">figure 2A</a>) were transfected with siGFP and siOPN. At 24 h posttransfection, approximately 3×10⁵ cells were seeded in transwell chamber for 48 h and images of invaded cells were recorded under microscope at least three individual fields per well at 10× magnification. (B) The invaded cells were counted in at least three individual fields per insert and represented by bar diagrams. The results shown are representative of two independent experiments performed in duplicate. (C) The above invaded cells were quantified in 96 wells plate at OD 560 nm using extraction buffer. Data represent means ± SD of two independent experiments performed in duplicate. *denotes p<0.05 compared to mock-infected Huh7.5 cells. **denotes p<0.05 compared to HCV-infected cells transfected with siGFP.</p

Colocalization of OPN with integrin αVβ3 and CD44.

<p>(A, B) Mock and HCV-infected cells (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087464#pone-0087464-g002" target="_blank">figure 2A</a>) were transfected with siGFP and siOPN. At 72 h posttransfection, cells were permeabilized and incubated with anti-OPN, anti-αVβ3, anti-CD44 and anti-HCV NS5A antibodies for 1 h at RT, followed by incubation with secondary antibodies; for OPN (anti-goat Alexa Fluor 546), αVβ3 (anti-mouse Alexa Fluor 488), CD44 (anti-mouse Alexa Fluor 488) and HCV NS5A (anti-rabbit Alexa Fluor 633). DAPI was used as a nuclear stain. Arrows represent colocalization of OPN with αVβ3 and CD44 respectively. HCV NS5A represents HCV infection. Scale bar 10 µM. (C) Colocalization of OPN with pan-cadherin (plasma membrane marker). Mock and HCV-infected cells (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087464#pone-0087464-g002" target="_blank">figure 2A</a>) were permeabilized and incubated with anti-OPN, anti-pan-cadherin and anti-HCV NS5A antibodies for 1 h at RT, followed by incubation with secondary antibodies; for OPN (anti-goat Alexa Fluor 546), pan-cadherin (anti-rabbit Alexa Fluor 488) and HCV NS5A (anti-rabbit Alexa Fluor 633). (D) Similarly, non-permeabilized mock and HCV-infected cells were incubated with anti-OPN and anti-pan-cadherin antibodies for 1 h at RT and then cells were permeabilized and incubated with anti-HCV NS5A antibody for 1 h at RT followed by 1 h incubation with above secondary antibodies. DAPI was used as a nuclear stain. Arrows represent colocalization of OPN with pan-cadherin. (E) Colocalization of OPN with PDI (ER marker). As described in panel C and D, permeabilized cells were incubated with anti-OPN, anti-PDI and anti-HCV NS5A antibodies for 1 h at RT, followed by incubation with secondary antibodies; for OPN (anti-goat Alexa Fluor 546), PDI (anti-rabbit Alexa Fluor 488) and HCV NS5A (anti-rabbit Alexa Fluor 633). (F) Simultaneously, non-permeabilized cells were incubated with anti-OPN and anti-PDI antibodies for 1 h at RT and then cells were permeabilized and incubated with anti-HCV NS5A antibody for 1 h at RT followed by 1 h incubation with above secondary antibodies. DAPI was used as a nuclear stain. Arrows represent colocalization of OPN with ER marker. HCV NS5A represents HCV infection. Scale bar 10 µM.</p

Interaction of OPN with integrin αVβ3 and CD44.

<p>HCV-infected Huh7.5 cells were transfected with siGFP and siOPN as described in Materials and Methods. At 72 h posttransfection, equal amounts of cellular lysates from mock, HCV-infected cells and HCV-infected cells transfected with above siRNA were immunoprecipitated using anti-OPN (1∶100) antibody and immunoblotted with anti-integrin β3 and anti-CD44 (lane 1–4). Similarly the cellular lysates from HCV-infected cells were immunoprecipitated with anti-OPN and isotype control goat IgG antibodies in two different sets followed by immunoblot analysis using anti-β3 and anti-CD44 (lane 5–8).</p

HCV induces hepatoma cells migration.

<p>(A) HCV-infected cells (from the same pool of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087464#pone-0087464-g002" target="_blank">figure 2A</a>) were transfected with siGFP and siOPN and migration was examined by wound healing assay. Images were taken at 0 h and 48 h postwounding. Arrows indicate the wound of monolayer cells scratched using pipette tips. The results shown are representative of three independent experiments. (B) The percent migrated depth of above cells was measured in three independent experiments represented by bar diagram. *denotes p<0.05 compared to mock-infected Huh7.5 cells. **denotes p<0.05 compared to HCV-infected cells transfected with siGFP.</p

Role of HCV-induced OPN in cell signaling cascade.

<p>(A) HCV-infected cells were transfected with siGFP, siOPN, siCD44, and siβ3 as described in Materials and Methods. At 72 h posttransfection, cells were harvested and equal amount of cellular lysates were subjected to western blot analysis using anti-OPN, anti-β3 and anti-CD44. (B) The above cellular lysates were subjected to western blot analysis using anti-FAK, anti-p-Akt (Ser ⁴⁷³), anti-p-Src (Tyr ⁴¹⁶), and anti-N-cadherin antibodies. HCV NS3 was used as a representative of HCV-infection. (C) Mock (Huh7.5) cells were treated with recombinant human OPN (rhOPN) (50 nM) for 48 h. Cells were harvested and equal amounts of cellular lysates were immunoblotted using anti-FAK and anti-p-Akt (Ser⁴⁷³) (lane 1, 2), anti-E-cadherin, anti-N-cadherin, anti-pSrc ⁴¹⁶ (lane 3, 4). (D) Cellular lysates from HepG2 cells incubated with cell culture supernatants from mock, HCV-infected cells and those infected cells transfected with siOPN and siGFP were immunoblotted using anti-E-cadherin and anti-N-cadherin (lane 1–4). Similarly cellular lysates from HepG2 cells incubated with cell culture supernatants from mock and HCV-infected cells with/without immunodepletion by anti-OPN, were immunoblotted using anti-E-cadherin and anti-N-cadherin (lane 5–7). Immunodepletion by isotype control goat IgG antibody was used as control (lane 8). Actin was used as protein loading control. The results shown are the representative of three independent experiments.</p