Investigation of the roles of ERK, JNK, and PKC in the regulation of miR-21 expression upon HCV infection.

<p>(A) Huh7 cells were co-transfected with miPPR21 and pCMV-NS5A (<i>left panel</i>) or pCMV-NS3/4A (<i>right panel</i>) for 24 h, and then signal pathway specific inhibitors (20 µM each) were then added, as indicated. The cells were lysed and luciferase activity was measured. (B) Cells were transfected with pCMV-NS5A (<i>left panel</i>) or pCMV-NS3/4A (right panel) for 24 h, and then treated with the signal pathway inhibitors (20 µM each) as indicated. The phosphorylation and total protein levels of c-Jun (<i>left panel</i>) and c-Fos (<i>right panel</i>) were determined by Western blot (<i>upper panel</i>), and miR-21 expression was measured by qPCR (<i>lower panel</i>). (C) Huh7 cells were co-transfected with miPPR21 and dominant-negative mutants of ERK1 (mERK1), ERK2 (mERK2), JNK (mJNK) or control vectors at different concentrations, as indicated and the resultant luciferase activities were measured. All experiments were repeated at least three times with similar results. Bar graphs represent the means ± SD, n = 3.</p

Determination of the expression of miR-21 during HCV infection in hepatocytes.

<p>(<i>A</i>) Human Huh7 hepatocytes were infected with or without HCV (MOI = 1) for different times as indicated. The expression of miR-21 was measured by qPCR and normalized to the expression of U6 in each sample. Results are standardized to 1 in uninfected cells. (<i>B</i>) Human Huh7 hepatocytes were infected with or without HCV at different MOIs, as indicated, for 12 h, and expression miR-21 was determined by qPCR. Data are presented as the meansSD (n = 3) from one representative experiment. Similar results were obtained in three independent experiments.</p

miR-21 regulates components of the Toll-like receptor 7 signaling cascade.

<p>Huh7 hepatocytes were transfected with miR-21 mimics or control RNA, miR-21 inhibitor or control inhibitor (final concentration, 50 nM). After 48 h, MyD88, IRAK1, IRAK4, and TRAF6 mRNA levels were determined by qPCR (<i>A</i>, <i>B</i>, <i>C</i>, and <i>D</i>) and RT-PCR (<i>E</i> and <i>F</i>), respectively. Data are presented as the meansSD (n = 3) from one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.</p

miR-21 suppresses HCV-triggered type I IFN production.

<p>(A) Human Huh7 hepatocytes (0.5 ml, 2×10⁵ cells) were transfected with control miRNA (miR-ctrl) or miR-21 mimics (<i>left</i>), and control inhibitor (ctrl) or miR-21 inhibitor (<i>right</i>), as indicated at a final concentration of 50 nM. RNA was isolated 48 h post-transfection, and miR-21 expression was measured by qPCR and normalized to U6 snRNA. Results are standardized to 1 in control cells. (B) Huh7 hepatocytes were infected with or without HCV (MOI = 1) for different times as indicated. IFN-α mRNA levels (<i>left</i>) were determined by qPCR and normalized to the expression of GAPDH in each sample. IFN-α secretion into the cell culture medium (<i>right</i>) was measured by ELISA. (<i>C</i> and <i>D</i>) Huh7 hepatocytes were transfected with miR-21 mimics or control RNA (<i>C</i>), miR-21 inhibitor or control inhibitor (<i>D</i>), as indicated. After 48 h, the cells were transfected with FL-J6/JFH5′C19Rluc2AUbi (0.1 µg) for the indicated time. IFN-α mRNA levels were determined by qPCR, and IFN-α secretion into the medium was measured by ELISA. Data are shown as the meansSD (n = 3) from one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.</p

Proposed model for the activation of miR-21 expression upon HCV infection and the role of miR-21 in the regulation of the antiviral activity of type I IFN.

<p>During HCV infection, the virus is first recognized by TLRs and RIG-1, which in turn activates MyD88 and IRAK1 to initiate IFN-α synthesis, resulting in the activation of ISGs and the inhibition of HCV replication. In addition, miR-21 expression is activated during HCV infection through two signaling pathways: the PKCε/JNK/c-Jun pathway and the PKCα/ERK/c-Fos pathway. The HCV NS5A protein activates PKCε to enhance the expression of JNK and c-Jun, while the HCV NS3/4A complex stimulates PKCα to promote the production of ERK and c-Fos. The two subunits (c-Jun and c-Fos) of AP-1 join together to recognize the miR-21 promoter and activate the expression of miR-21, which represses the expression of MyD88 and IRAK1 via imperfect base pairing between miR-21 and the 3′UTR of MyD88 and IRAK1. The reduction in MyD88 and IRAK1 causes a reduction of type-I IFN production and ISG expression that might contribute to viral pathogenesis and virus propagation.</p

Functional analysis of <i>cis</i>-regulatory elements involved in the activation of miR-21 regulated by HCV proteins.

<p>(A) Huh7 cells were co-transfected with miR-21-luciferase reporter plasmid (miPPR-21) and the plasmids encoding each of the 11 HCV proteins, as indicated, for 24 h. The activity of the miR-21 promoter was measured by luciferase activity assays (<i>left panel</i>), and the levels of miR-21 expression were determined by qPCR (<i>right panel</i>). (B) The consensus sequences of the miR-21 promoter region (−410 to +38) in the reporter plasmid miPPR-21. Conserved bases across vertebrates are shown in capitals and nonconserved bases or deletions are denoted by “n”. The potential <i>cis</i>-acting elements for transcription factors are indicated. (C) Promoter analysis by transient expression of a series of reporter plasmids of miPPR-21 (intact, f1; truncated, f2–f4; mutated, f5–f14) in Huh7 cells. Data are presented as the meansSD from three experiments. (D) Huh7 cells were transfected with pCMV-NS5A or pCMV-NS3/4A at different time intervals, respectively. The phosphorylation and total protein levels of c-Jun and c-Fos were determined by Western blot. (E) AP-1 binding sites were determined by ChIP assays. All experiments were repeated at least three times with similar results.</p

miR-21 targets human MyD88 and IRAK1.

<p>(<i>A</i>) Sequence alignment of miR-21 and its binding sites in the 3′ UTRs of MyD88 and IRAK1, as predicted by RNA22 software. (<i>B</i>) Huh7 hepatocytes (1×10⁴) were co-transfected with pGL3-Basic, pGL3-MyD88 3′ UTR, or pGL3-IRAK1 3′ UTR firefly luciferase reporter plasmids (80 ng) and pRL-TK <i>Renilla</i> luciferase plasmid (40 ng), together with miR-21 mimics or control RNA, miR-21 inhibitor or control inhibitor (final concentration, 50 nM), as indicated. After 48 h, firefly luciferase activity was determined and normalized to <i>Renilla</i> luciferase activity. (<i>C</i>) HEK293 cells (1×10⁴) were co-transfected with GFP control, GFP-MyD88 3′ UTR, or GFP-IRAK1 3′ UTR plasmid (400 ng), together with miR-21 mimics or control RNA (final concentration, 50 nM), as indicated. After 48 h, GFP expression was analyzed by FACS, and the mean fluorescence intensity (MFI) of GFP was determined. (<i>D</i> and <i>E</i>) Huh7 hepatocytes (1×10⁶) were transfected with miR-21 mimics (<i>D</i>) or miR-21 inhibitor (<i>E</i>) at various concentrations for 48 h (<i>left</i>), or at 50 nM (final concentration) for the indicated time (<i>right</i>). MyD88 and IRAK1 protein levels were determined by Western blot and normalized to β-actin (<i>top panel</i>); MyD88 and IRAK1 mRNA levels were determined by qPCR and normalized to GAPDH (<i>bottom panel</i>). Data are presented as the meansSD (n = 3) from one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.</p

Involvement of PKCε and PKCα in the induction of miR-21.

<p>(<i>A and E</i>) Time-dependent JNK (<i>A</i>) and ERK (<i>E</i>) phosphorylation determined by Western blot. (<i>B and F</i>) Huh7 cells were co-transfected with miPPR-21 and pCMV-NS5A (<i>B</i>) or pCMV-NS3/4A (<i>F</i>) along with siRNA expression plasmids against different isoforms of PKC or siCtrl. A similar vector (pSilencer 2.0) containing an irrelevant sequence that does not show significant homology to any human gene was provided by Ambion, Inc. and used as a negative control. Luciferase activities were measured. (<i>C and G</i>) Cells were transfected with pCMV-NS3/4A (<i>C</i>) or pCMV-NS5A (<i>G</i>) and treated with PKC siRNA expression plasmids or si-Ctrl, as indicated. The phosphorylation and total protein levels of c-Jun (<i>C</i>) and c-Fos (<i>G</i>) were determined by Western blot (upper panel), and miR-21 expression was measured by qPCR (lower paned). (<i>D</i>) Inhibition of p-JNK by SP600125, GF109203, and PKCε-specific siRNA (PKCε siRNA) in the presence of NS3/4A presented. (<i>H</i>) U0126, GF109203, and PKCα-specific siRNA (PKCα siRNA) attenuated ERK phosphorylation when treated with NS5A. All experiments were repeated at least three times with similar results. Bar graphs represent means ± SD, n = 3.</p

miR-21 stimulates HCV replication and attenuates the HCV response to IFN-α treatment.

<p>(<i>A</i>) Huh7 hepatocytes were transfected with control RNA (miR-ctrl) or miR-21 mimics (final concentration, 50 nM). After 48 h, cells were infected with HCV (MOI = 1) for 2 h and washed before fresh medium was added. After 72 h, intracellular HCV RNA replicates were quantified by qPCR and normalized to the GAPDH internal control. (<i>B</i>) Huh7 hepatocytes were transfected as described in (<i>A</i>) and infected with HCV (MOI = 1) for 2 h. After 48 h, HCV core expression was analyzed by Western blot (top panel) using β-actin as a loading control (bottom panel). (<i>C</i>) Huh7 hepatocytes were transfected with miR-21 mimics or control RNA (miR-ctrl) (final concentration, 50 nM). After 48 h, cells were infected with HCV (MOI = 1) for 2 h and washed before adding fresh medium with or without recombinant human IFN-α (100 U/ml). After 72 h, intracellular HCV RNA replicates were quantified by qPCR. (<i>D and E</i>) Huh7 hepatocytes were transfected with miR-21 inhibitors or control inhibitor (ctrl) (final concentration, 50 nM). After 48 h, cells were infected with HCV (MOI = 1) for 2 h and washed before adding fresh medium with or without recombinant human IFN-α (100 U/ml) or anti-IFN-α-neutralizing antibody (100 neutralizing units/ml) as indicated. After 72 h, RNA was isolated from the cell culture medium, and supernatant HCV replicates (<i>D</i>) were measured by qPCR. Intracellular HCV RNA replicates (<i>E</i>) were quantified by qPCR using GAPDH as internal control. Data are presented as the meansSD (n = 3) from one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.</p

Regulation of IFN signaling by miR-21 is achieved primarily through MyD88 and IRAK1.

<p>(<i>A</i>) Huh7 hepatocytes were transfected with nonspecific control siRNA or siRNA against MyD88 or IRAK1, as indicated. After 24 h, MyD88 and IRAK1 mRNA levels were determined by qPCR and normalized to GAPDH (<i>lower panel</i>); after 48 h, MyD88 and IRAK1 protein levels were determined by Western blot and normalized to β-actin (<i>upper panel</i>). (<i>B</i>) Huh7 hepatocytes were co-transfected with miR-21 mimic or control RNA and siRNA against MyD88 or IRAK1, as indicated. After 48 h, Huh7 cells were transfected with FL-J6/JFH5′C19Rluc2AUbi (0.1 µg) for the indicated time, and IFN-α expression and secretion were determined by qPCR and ELISA, respectively. (<i>C</i>) Huh7 hepatocytes were co-transfected with miR-21 inhibitor or control inhibitor and siRNA against MyD88 or IRAK1 or nonspecific control siRNA, as indicated. After 48 h, Huh7 cells were transfected with FL-J6/JFH5′C19Rluc2AUbi (0.1 µg) for the indicated times, and IFN-α expression and secretion were determined by qPCR and ELISA, respectively. Data are the meansSD (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.</p