Repression of MicroRNA-30e by Hepatitis C Virus Enhances Fatty Acid Synthesis

Cell Culture, HCV Infection, and Transfection

Huh7.5 cells and Huh7.5 cells harboring the HCV genotype 2a genome-length replicon (Rep2a cells; kindly provided by Hengli Tang, Florida State University, FL, USA) were used. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10 % fetal bovine serum and penicillin–streptomycin at 37°C in a 5% CO₂ atmosphere.

HCV genotype 2a (clone JFH1) was grown in Huh7.5 cells as described.15 Virus released into cell culture supernatant was filtered through a 0.45-μm pore cellulose acetate membrane and quantitated in standard IU/mL. For infection, cells were incubated with HCV genotype 2a (clone JFH1) (multiplicity of infection, 0.1) in a minimum volume of medium as described.16 The cellular RNA was extracted 3 days postinfection.

Transfection of ATG5 small interfering RNA (siRNA) into Huh7.5 cells was performed using lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA). Briefly, Huh7.5 cells were plated at a density of approximately 1 × 10⁵ cells/well in a 12-well plate and transfected with 50 nM ATG5 siRNA (sc-41445; Santa Cruz, Dallas, TX) or control siRNA and lysed for western blot analysis at 48 hours after transfection. Transfection of an miR-30e-5p mimic (002223; ThermoFisher Scientific, Waltham, MA) into Huh7.5 cells was performed using lipofectamine (Invitrogen). Briefly, Huh7.5 cells were plated at a density of approximately 1 × 10⁵ cells/well in a 12-well plate and transfected with 20 nM of miR-30e-5p mimic or control miR. Cells were lysed for western blot analysis 48 hours after transfection. Total RNA was prepared in a separate transfection.

RNA Quantitation and Reverse-Transcription Real-Time Polymerase Chain Reaction

Total RNA was isolated by using a TRIzol reagent (Invitrogen). RNA was quantified by using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Complementary DNA was synthesized using miR-30e- or a U6-specific primer (Thermo Fisher Scientific) with a TaqMan miRNA reverse transcription (RT) kit or random hexamers and a Superscript III RT kit (Invitrogen). Real-time polymerase chain reaction (qPCR) was performed with a 7500 real-time PCR system (Applied Biosystems, Foster City, CA). TaqMan universal PCR master mix and a 6-carboxyfluorescein (FAM)-minor groove binder (MGB) probe for ATG5 (Hs00169468_m1; Thermo Fisher Scientific) and 18S ribosomal RNA (rRNA; Hs03928985_g1; Thermo Fisher Scientific) were used. Relative expression level was calculated by normalizing with U6 or 18S rRNA, using the 2^−ΔΔCT formula (^ΔΔCT = ^ΔCT of the sample − ^ΔCT of the untreated control).

Western Blot Analysis

Cells were lysed by using a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer. The lysates were subjected to PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% nonfat dried milk and probed with the following specific primary antibodies: C/EBP-β, sterol regulatory element binding protein (SREBP)-1 (2A4), and fatty acid synthase (FASN) (A-5; Santa Cruz); ATG5 (Proteintech, IL); and mammalian target of rapamycin (mTOR; 7C10) and phosphorylated-mTOR (p-mTOR; D9C2) (Cell Signaling Technology, Danvers, MA). After washing, the blots were incubated with secondary antibody for 1 hour. Proteins were detected by using an enhanced chemiluminescence western blot substrate (Pierce; ThermoFisher Scientific). Membranes were reprobed with antibody to actin (Santa Cruz) as an internal control for normalization of the protein load. ImageJ software (National Institutes of Health [NIH]) was used for densitometric scanning of western blot images.

Luciferase Reporter Assays

The miR-30e promoter (nucleotides −1813 to +1; P0) or promoter fragment of the deletion mutant of miR-30e (P1) were PCR amplified from Huh7.5 genomic DNA by using specific primers (Table 1). The fragments were digested with MluI and HindIII and cloned into the pGL3-Basic luciferase vector (Promega, Madison, WI). Huh7.5 cells and Rep2a cells were transfected with a luciferase reporter plasmid containing the miR-30e promoter (1 µg/mL) by using Lipofectamine 3000 (Invitrogen). Luciferase activity was determined as described.17

Huh7.5 cells were plated at a density of 0.8 × 10⁵ cells/well in a 24-well plate and cotransfected with 25 nM C/EBP-β siRNA (Santa Cruz) or control siRNA and the promoter construct of miR-30e using Endofectin (GeneCopoeia, Rockville, MD). A luciferase reporter assay was performed 48 hours after transfection.

Ago2-RNA Co-Immunoprecipitation

The hepatocyte RNA–miR-30e complex was precipitated as described.18 Briefly, miR-30e transfected Huh7.5 cells were lysed with lysis buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM ethylene diamine tetraacetic acid, 1% Triton X-100, 5 mM dithiothreitol, protease inhibitor mixture, and 100 U/mL RNase-OUT [Invitrogen]). Lysates were clarified, incubated with an anti-Ago2 monoclonal antibody (11A9; Sigma-Aldrich, St. Louis, MO) or isotype control immunoglobulin (Ig)G2a at 4°C for 7 hours, and mixed with protein G-Sepharose (GE Healthcare, Piscataway, NJ) overnight. The beads were washed 4 times, and RNA was isolated by using TRIzol reagent (Invitrogen). Target RNA was quantitated by RT-qPCR using specific primers.

Immunofluorescence

Huh7.5 cells were transfected with miR-30e mimic and infected with green fluorescent protein (GFP)-tagged HCV. Cells were fixed for 30 minutes with 4% paraformaldehyde after 72 hours of infection and then treated with 0.2% Triton X-100 for 5  minutes. After blocking with 3% bovine serum albumin, cells were incubated with primary antibody rabbit polyclonal microtubule-associated protein light chain 3B (LC3B; Sigma-Aldrich) and then conjugated to Alexa Fluor 594 (Molecular Probes, Eugene, OR) with anti-rabbit Ig. 4´,6-Diamidino-2-phenylindole (DAPI) was incubated for nuclear staining. Three-channel optical images (green for HCV, red for LC3, and blue for nucleus) were collected using the sequential scanning mode of the Olympus FV1000 confocal system (Olympus, Center Valley, PA). Images were superimposed digitally for fine comparisons.

Huh7.5 cells were transfected with ATG5 siRNA (sc-41445; Santa Cruz) or control siRNA for 48 hours. Cells were stained by boron-dipyrromethene (BODIPY) 493/503 (Invitrogen) for 1 hour, and DAPI was incubated for nuclear staining. Lipid accumulation was observed by using the Olympus FV1000 confocal system (Olympus) in cells with DAPI-stained nuclei. Images were superimposed digitally for fine comparisons. ImageJ software (NIH) was used for densitometric scanning of immunofluorescence images.

Statistical Analysis

Results are presented as mean ± SD. Data were analyzed by the Student t test with a two-tailed distribution. P < 0.05 was considered statistically significant.

HCV Infection Down-Regulates miR-30e Expression

We have previously shown that miR-30e expression was significantly lower (P < 0.003) in the sera of patients with HCV-infected HCC compared with healthy volunteers.19 We also observed down-regulation of miR-30e in liver samples from patients with HCV-associated HCC, although the mechanism of miR-30e down-regulation remains unknown. We examined the expression level of miR-30e in HCV-infected human hepatocytes. The level of miR-30e was significantly reduced following HCV infection in Huh7.5 cells (Fig. 1, panel A). HCV RNA was measured from HCV-infected Huh7.5 cells by RT-qPCR (Fig. 1, panel B). A similar result was obtained when we examined the level of miR-30e in Huh7.5 cells harboring the genome-length HCV replicon (Rep2a cells) compared to parental Huh7.5 cells (Fig. 1, panel C). Together, our results indicate that HCV infection suppresses miR-30e expression in hepatocytes.

HCV Infection Down-Regulates C/EBP-β-Mediated miR-30e Promoter Activation

We next examined how HCV modulates miR-30e expression. For this, an miR-30e promoter sequence (nucleotides −1,813 to +1; P₀) was cloned into pGL3 luciferase reporter plasmid DNA, using specific primers (Table 1). Huh7.5 cells and Huh7.5 cells harboring the HCV genome-length replicon cells were transfected with the P₀ construct to examine promoter activity. miR-30e promoter activity was significantly down-regulated in HCV replicon-expressing cells compared to control parental cells (Fig. 2, panel A). To determine the cis-regulatory elements present in the miR-30e promoter region (nucleotides −1,813 to +1), we searched PROMO, using version 8.3 of TRANSFAC. Several regulatory elements were found in the designated consensus sequence of the miR-30e promoter, including binding sites for C/EBP-β. C/EBP-β, which belongs to the leucine zipper family, is an important transcriptional regulator.20 Moreover, we have previously shown that HCV down-regulates promoter activity of complement C3 and miR-181c by C/EBP-β.21, 22

We next identified the specific region on the miR-30e promoter responsible for HCV-mediated repression. We generated deletion mutants of the miR-30e promoter constructs (nucleotides −1567 to +1 [P₁]) and cloned these into pGL3 luciferase reporter plasmid DNA (Fig. 2, panel B). We knocked down C/EBP-β using a specific siRNA and examined the activity of the P₀ or P₁ construct of the miR-30e promoter in Huh7.5 cells to analyze the role of C/EBP-β in the regulation of HCV-mediated miR-30e expression. Knockdown of C/EBP-β inhibited the activity of the miR-30e P₀ promoter, but the P₁ fragment did not exert an effect (Fig. 2, panel C), suggesting that the trans-suppressor element may be located between regions –1,813 to –1,565. We further confirmed that HCV infection in Huh7.5 cells reduces C/EBP-β expression (Fig. 2, panel D), in agreement with previous reports.21, 22 Transfection of siRNA to C/EBP-β in Huh7.5 cells reduced protein expression (Fig. 2, panel E). Together, these results suggest that HCV-mediated C/EBP-β suppression is an important mechanism for miR-30e down-regulation.

ATG5 IS a Direct Target of miR-30e

In silico prediction suggests that ATG5 is a direct target of miR-30e (Fig. 3, panel A). A recent report showed that miR-30e targets the 3′-untranslated region (UTR) of ATG5 and inhibits its translation in gastric cancer cells.23 We examined the interaction between miR-30e and ATG5 in hepatocytes. For this, miR-30e-transfected Huh7.5 cell lysates were immunoprecipitated with an Ago2-specific monoclonal antibody or an unrelated isotype control. RNA was isolated from the immunoprecipitated lysates and analyzed by RT-qPCR. Higher ATG5 level was noted in the miR-30e–Ago2 complex compared to immunoprecipitated control, suggesting its presence in their complex (Fig. 3, panel B). Overexpression of miR-30e inhibits ATG5 expression (Fig. 3, panel C). siRNA to ATG5 was used as a positive control. As reported,23 we did not observe the inhibition of ATG5 mRNA by miR-30e in our experimental system. We and others have shown that HCV induces autophagy.17, 24 ATG5 is an important molecule in the autophagy signaling pathway. We therefore predicted that overexpression of miR-30e in HCV-infected hepatocytes will reduce ATG5 expression and may impair the induction of autophagy. For this, we overexpressed miR-30e in GFP-tagged HCV-infected cells and stained these for LC3, a marker for autophagy. Our results demonstrated that overexpression of miR-30e inhibited HCV-induced LC3 lipidation compared to control miR-treated cells (Fig. 3, panel D). We also observed that overexpression of miR-30e inhibited HCV-induced LC3 lipidation by western blot analysis (Fig. 3, panel E).

Depletion of ATG5 Inhibits Lipid Synthesis in Hepatocytes

LDs have been implicated as an essential link for efficient autophagy in the yeast system,25 and autophagy is inhibited following depletion of free fatty acids. In HCV infection, both autophagy and LDs are induced in hepatocytes. Because ATG5 is a direct target of miR-30e, we wanted to understand the interplay between ATG5 and lipid synthesis. ATG5 and ATG7 are essential molecules for the induction of autophagy; ATG7 conjugates ATG5 to ATG12 for autophagosome formation.26 ATG7-deficient mice suppress LD formation,27 and SREBP-1c forms LDs through de novo lipogenesis.28 To investigate the relationship between ATG5 and SREBP-1c expression, we knocked down ATG5 using a specific siRNA and performed western blot analysis using specific antibodies. A significant down-regulation of SREBP-1c and FASN expression level was observed compared to that of the control cell after normalization with actin (Fig. 4, panel A). A similar result was obtained when we examined the expression of SREBP-1c and FASN in Huh7.5 cells harboring the genome-length HCV replicon (Rep2a cells) compared to parental cells (Fig. 4, panel B). We also observed a significant down-regulation of p-mTOR, an intermediate between ATG5 and SREBP-1c expression level, compared to that of the control cell (Fig. 4, panel C). We further showed an inhibition of LDs in ATG5-depleted cells (Fig. 4, panel D). Together, these data suggest that depletion of ATG5 inhibits SREBP-1c and FASN expression.

Overexpression of miR-30e Inhibits Lipid Synthesis in Hepatocytes

SREBP-1 and FASN are important players in lipid metabolism, and SREBPs are key transcriptional factors in lipogenesis and lipid uptake.29, 30 We observed that HCV infection in Huh7.5 cells increased expression of SREBP-1c and its downstream target FASN (Fig. 5, panel A). We also observed ATG5 depletion inhibits SREBP-1c and FASN expression. We therefore examined whether miR-30e overexpression modulates lipid synthesis in hepatocytes. For this, we performed western blot analysis in Huh7 cells transfected with an miR-30e mimic or control miR. A significant down-regulation of SREBP-1c and FASN expression level compared to that of the control parental cell was observed after normalization with the housekeeping protein actin (Fig. 5, panel B). A similar result was obtained when we examined the expression of SREBP-1c and FASN in Huh7.5 cells harboring the genome-length HCV replicon (Rep2a cells) compared to parental cells (Fig. 5, panel C). Together, our data suggest that mir-30e inhibits ATG5 and eventually SREBP-1c and FASN in hepatocytes.