MiR-942 Mediates Hepatitis C Virus-Induced Apoptosis via Regulation of ISG12a

<div><p>The interaction between hepatitis C virus (HCV) and human hepatic innate antiviral responses is unclear. The aim of this study was to examine how human hepatocytes respond to HCV infection. An infectious HCV isolate, JFH1, was used to infect a newly established human hepatoma cell line HLCZ01. Viral RNA or NS5A protein was examined by real-time PCR or immunofluorescence respectively. The mechanisms of HCV-induced IFN-β and apoptosis were explored. Our data showed that HLCZ01 cells supported the entire HCV lifecycle and IFN-β and interferon-stimulated genes (ISGs) were induced in HCV-infected cells. Viral infection caused apoptosis of HLCZ01 cells. Silencing of RIG-I, IRF3 or TRAIL inhibited ISG12a expression and blocked apoptosis of viral-infected HLCZ01 cells. Knockdown ISG12a blocked apoptosis of viral-infected cells. MiR-942 is a candidate negative regulator of ISG12a predicted by bioinformatics search. Moreover, HCV infection decreased miR-942 expression in HLCZ01 cells and miR-942 was inversely correlated with ISG12a expression in both HCV-infected cells and liver biopsies. MiR-942 forced expression in HLCZ01 cells decreased ISG12a expression and subsequently suppressed apoptosis triggered by HCV infection. Conversely, silencing of miR-942 expression by anti-miR-942 increased ISG12a expression and enhanced apoptosis in HCV-infected cells. Induction of Noxa by HCV infection contributed to ISG12a-mediated apoptosis. All the data indicated that innate host response is intact in HCV-infected hepatocytes. MiR-942 regulates HCV-induced apoptosis of human hepatocytes by targeting ISG12a. Our study provides a novel mechanism by which human hepatocytes respond to HCV infection.</p></div

Targeting Chemophotothermal Therapy of Hepatoma by Gold Nanorods/Graphene Oxide Core/Shell Nanocomposites

Nanographene oxide (NGO) are highly suitable to be the shells of inorganic nanomaterials to enhance their biocompatibility and hydrophilicity for biomedical applications while retaining their useful photonic, magnetic, or radiological functions. In this study, a novel nanostructure with gold nanorods (AuNRs) encapsulated in NGO shells is developed to be an ultraefficient chemophotothermal cancer therapy agent. The NGO shells decrease the toxicity of surfactant-coated AuNRs and provide anchor points for the conjugation of hyaluronic acid (HA). The HA-conjugated NGO-enwrapped AuNR nanocomposites (NGOHA-AuNRs) perform higher photothermal efficiency than AuNRs and have the capability of targeting hepatoma Huh-7 cells. NGOHA-AuNR is applied to load doxorubicin (DOX), and it exhibits pH-responsive and near-infrared light-triggered drug-release properties. Chemophotothermal combined therapy by NGOHA-AuNRs-DOX performs 1.5-fold and 4-fold higher targeting cell death rates than single chemotherapy and photothermal therapy, respectively, with biosafety to nontargeting cells simultaneously. Furthermore, our strategy could be extended to constructing other NGO-encapsulated functional nanomaterial-based carrier systems

HCV infection of HLCZ01.

<p>(<b>A</b>) Filtered supernatant of JFH1 RNA-transfected Huh7.5 cells was inoculated with naive Huh7.5 or HLCZ01 cells. Cells were immunostained with mouse monoclonal anti-NS5A antibody at day 6 after inoculation. DAPI was used for nuclei counterstaining. (<b>B</b>) Naïve HLCZ01 cells were incubated for 3 days with filtered, conditioned media collected from HCV-infected HLCZ01 cells and immunostained for NS5A expression. (<b>C</b>) Viral RNA kinetics determined by real-time PCR in HLCZ01 and Huh7.5 cells infected by JFH1 virus at MOI of 0.1. HCV RNA in HCV-infected cells was determined by real-time PCR. The viral replication is represented by HCV genome equivalence (GE)/µg total cellular RNA. (<b>D</b>) Viral RNA in the supernatant of HCV-infected HLCZ01 and Huh7.5 cells determined by real-time PCR. The viral RNA is calculated as GE per milliliter medium using a standard curve generated by in vitro transcribed full-length JFH1 RNA. (<b>E</b>) IFN inhibits HCV RNA replication in HLCZ01 cells in a dose-dependent manner. (<b>F</b>) Anti-CD81 antibody blocked HCV infection in HLCZ01 cells. HLCZ01 cells were pretreated with anti-CD81 antibody for 2 hours before viral inoculation. Viral RNA was analyzed by real-time PCR at day 3 pi. If not stated otherwise bar graphs represent means of three independent experiments. Horizontal dashed lines indicate the low limit of quantification (LLOQ) of the assay.</p

Induction of IFN-β and apoptosis by HCV infection is mediated through RIG-I and IRF-3.

<p>(<b>A</b>) Confirmation of RIG-I knockdown with RIG-I-specific shRNA in HLCZ01 cells. RIG-I shRNA or control shRNA was delivered into HLCZ01 cells followed by HCV infection. RIG-I protein was determined by western blot. (<b>B</b>) Knocking down RIG-I inhibits the induction of IFN-β and ISG12a by HCV infection in HLCZ01 cells. HLCZ01 cells were treated as described in part A. The expression of IFN-β or ISG12a mRNA was examined using real-time PCR and normalized with GAPDH. (<b>C</b>) RIG-I knockdown blocks HCV-induced apoptosis of HLCZ01 cells. RIG-I shRNA or control shRNA was delivered into HLCZ01 cells followed by HCV infection for 9 days. Apoptosis of HLCZ01 cells was examined using flow cytometry. (<b>D</b>) IRF-3 directly regulates IFN-β and ISG12a mRNA expression in HCV-infected HLCZ01 cells. HLCZ01 cells were transfected with control shRNA or IRF-3–specific shRNA for 24 hours, followed by JFH-1 infection. The expression of IFN-β and ISG12a was detected using real-time PCR analysis and normalized with GAPDH. (<b>E</b>) Knockdown of IRF-3 reduces apoptosis of HCV-infected HLCZ01 cells. HLCZ01 cells were treated as described in part D. The cells were harvested for flow cytometry. (<b>F</b>) The knockdown efficiency was examined by western blot analysis using anti–IRF-3 antibody. The abbreviation “con” is for “control” in the figures. If not stated otherwise bar graphs represent means of three independent experiments.</p

Apoptosis induction by HCV infection in HLCZ01 cells involves ISG12a which relies on TRAIL-mediated pathway.

<p>(<b>A</b>) HCV infection induced TRAIL and its receptors DR4 and DR5. HLCZ01 cell were infected by HCV at MOI of 0.1 for different time periods. TRAIL, DR4 and DR5 mRNA was examined by real-time PCR and normalized with GAPDH. (<b>B/C</b>) Silencing of TRAIL inhibited the induction of ISG12a and blocked apoptosis of viral-infected cells. TRAIL shRNA was delivered into HLCZ01 cells, followed by HCV infection. (B) TRAIL and ISG12a mRNA was detected by real-time PCR and normalized with GAPDH. (C) Cells were collected for flow cytometry analysis. (<b>D</b>) Confirmation of ISG12a knockdown with ISG12a-specific shRNA in HLCZ01 cells. The plasmid pSilencer-ISG12a shRNA was delivered into HLCZ01 cells. ISG12a mRNA and protein was detected by real-time PCR and western blot receptively. (<b>E/F</b>) ISG12a knockdown prevented HCV-infected HLCZ01 cells from apoptosis. The plasmid pSilencer-ISG12a shRNA was delivered into HLCZ01 cells. The cells were infected with HCV for 9 days. PARP cleavage was examined by western blot (E). The cells were examined by flow cytometry (F). If not stated otherwise bar graphs represent means of three independent experiments.</p

Induction of Noxa contributes to ISG12a-mediated apoptosis.

<p>(<b>A</b>) Detection of cytosolic cytochrome c. HLCZ01 cells were infected with HCV at MOI of 0.1. Cytosolic cytochrome c was detected by western blot. (<b>B</b>) The cells were treated as described in part A. ISG12a, Noxa, Puma and Bax were detected by western blot. (<b>C</b>) Silencing of ISG12a or overexpression of miR-942 decreased Noxa expression. HLCZ01 cells were transfected by pcDNA3.1-ISG12a shRNA or pcDNA3.1-miR-942, followed with HCV infection at MOI of 0.1 for 9 days. Noxa and Puma were detected by western blot receptively. (<b>D</b>) Silencing of Noxa inhibited HCV-induced apoptosis. HLCZ01 cells were transfected by pcDNA3.1-Noxa shRNA or control vector, followed with HCV infection at MOI of 0.1 for 9 days. Noxa and PARP cleavage was detected by western blot. (<b>E</b>) Noxa overexpression reversed the inhibition of apoptosis of ISG12a-silenced HLCZ01 cells. HLCZ01 cells were transfected by ISG12a shRNA or control vector, followed with pcDNA3.1-Noxa transfection. Then the cells were infected with HCV at MOI of 0.1 for 9 days. The cells were collected and PARP cleavage was detected by western blot. If not stated otherwise blots are representative of three independent experiments.</p

HCV infection triggers apoptosis of HLCZ01 cells.

<p>(<b>A</b>) Annexin V expression determined by flow cytometry. HLCZ01 and Huh7.5 cells were infected with HCV at MOI of 0.1. The cells were harvested at day 9 pi and subjected to Annexin V analysis determined by Flow cytometry. The data are one representative of three independent experiments. (<b>B</b>) Kinetics of apoptosis in HCV-infected HLCZ01 cells. HCV-infected HLCZ01 and Huh7.5 cells were harvested for Annexin V expression determined by flow cytometry. The percentage of apoptotic cells is plotted. The data represent the means of three experiments. (<b>C</b>) Confirmation of HCV-induced apoptosis in HLCZ01 cells by western blot. HLCZ01 cells were infected with HCV at MOI of 0.1. Cells were collected and PARP cleavage was detected by western blot. Blots are representative of three independent experiments. (<b>D</b>) Blocking viral entry by anti-CD81 antibody or suppression of HCV replication by IFN reduces apoptosis of HLCZ01 cells. HLCZ01 cells were treated with anti-CD81 antibody or 100 IU/mL IFN before viral inoculation. The cells were harvested at day 9 pi for Annexin V expression determined by Flow cytometry. The graph shows the percentage of apoptotic cells, which represents the mean of 3 independent experiments.</p

HCV infection induces type I IFN and ISGs in HLCZ01 cells.

<p>(<b>A</b>) Kinetics of IFN-β in HCV-infected HLCZ01 cells. HLCZ01 and Huh7.5 cells were infected with JFH1 virus at MOI of 0.1. Cells were harvested for total RNA extraction at different time points. The kinetics of induction of IFN-β was analyzed by real-time PCR and normalized with GAPDH. (<b>B</b>) Kinetics of ISG12a, G1P3 and 1–8U in viral-infected HLCZ01 cells. HLCZ01 and Huh7.5 cells were treated as described in part A. The expression of ISG12a, G1P3 and 1–8U mRNA was analyzed by real-time PCR and normalized with GAPDH respectively. If not stated otherwise bar graphs represent means of three independent experiments.</p

Impact of ESCRT-II knockdown on dsRNA- or virus-induced chemokine expression.

<p><b>(A)</b> qPCR analysis of RANTES and IP-10 mRNA levels in PH5CH8 cells transfected with indicated siRNAs and mock-treated (empty bars), stimulated by 10 μg/ml of poly(I:C) for 6 h (grey bars), or infected with SeV at 160 HAU/ml for 8 h (black bars). <b>(B)</b> qPCR analysis of RANTES and IP-10 mRNA levels in Huh7.5-TLR3 cells transfected with indicated siRNAs and mock-treated (empty bars), stimulated by 10 μg/ml of poly(I:C) for 6 h (grey bars), or infected with HCV-JFH1 (MOI = 0.1) for 56 h (black bars). For the HCV groups cells were infected for 8 h prior to siRNA transfection for additional 48 h. “*” and “**” denote statistical differences exist as compared with control siRNA-transfected cells with a <i>P</i>-value of < 0.05 and < 0.01, respectively.</p

Pivotal role for the ESCRT-II complex subunit EAP30/SNF8 in IRF3-dependent innate antiviral defense

<div><p>The activation of interferon (IFN)-regulatory factor-3 (IRF3), characterized by phosphorylation and nuclear translocation of the latent transcription factor, is central to initiating innate antiviral responses. Whereas much has been learned about the upstream pathways and signaling mechanisms leading to IRF3 activation, how activated IRF3 operates in the nucleus to control transcription of IFNs remains obscure. Here we identify EAP30 (a.k.a, SNF8/VPS22), an endosomal sorting complex required for transport (ESCRT)-II subunit, as an essential factor controlling IRF3-dependent antiviral defense. Depletion of EAP30, but not other ESCRT-II subunits, compromised IRF3-dependent induction of type I and III IFNs, IFN-stimulated genes (ISGs) and chemokines by double-stranded RNA or viruses. EAP30, however, was dispensable for the induction of inflammatory mediators of strict NF-κB target. Significantly, knockdown of EAP30 also impaired the establishment of an antiviral state against vesicular stomatitis virus and hepatitis C virus, which are of distinct viral families. Mechanistically, EAP30 was not required for IRF3 activation but rather acted at a downstream step. Specifically, a fraction of EAP30 localized within the nucleus, where it formed a complex with IRF3 and its transcriptional co-activator, CREB-binding protein (CBP), in a virus-inducible manner. These interactions promoted IRF3 binding to target gene promoters such as IFN-β, IFN-λ1 and ISG56. Together, our data describe an unappreciated role for EAP30 in IRF3-dependent innate antiviral response in the nucleus.</p></div