HepG2 cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Life Technologies of Thermo Fisher Scientific, Waltham, MA, United States) at 37 °C in a humid atmosphere of 5% CO₂.

The HCV core fragment of genotype 1b (573 bp) was cloned from the template of a full-length HCV cDNA by PCR. The primers used for PCR amplification were as follows: forward 5’-ctcgagCCATGAGCACGAATCCTAAAC-3’ and reverse 5’-ggatccGGCGGAAACTGGGATGGT C-3’. The amplified product was digested with BamHI and XhoI and inserted into BamHI- and XhoI-digested pcDNA3.1/myc-His(-) vector.

The promoter region of SREBP2 (from nucleotide -933 to +263) was cloned from the genomic DNA of HepG2 cells using primers as follows: forward, 5’-gagctcTGATCCCATGGTATTCCATCG-3’; reverse, 5’-gctagcACCACTCACC GTCGATGTCT-3’. The PCR product was digested with SacI and NheI and inserted into SacI- and NheI-digested pGL4.10 vector (Promega, Madison, WI, United States) after sequencing.

To generate a luciferase reporter to determine the miRNA activity, the 3’-UTR (3596-4306; 776 bp) of SREBP2 was cloned by PCR using the following primers: forward, 5’-gctagcGCAGATGATTGTTAAGCTG-3’; reverse, 5’-ctcgagGGAAGGAACAGGACAATT-3’. The genomic DNA of HepG2 cells was used as the template. After digestion with NheI/XhoI, the PCR product was inserted into the pmirGLO reporter construct (Promega) at the NheI/XhoI restriction site after sequencing. Mutations of the seed region binding sites were generated by Pfu DNA polymerase (M7741; Promega) and DpnI (Promega). Three nucleotides in the 3’-UTR (binding for miR-185-5p) of the SREBP2 gene were mutated (T to G) by PCR. Wild-type and mutant constructs were confirmed by sequencing. The primers used for mutagenesis are shown in Table 1.

Determination of intracellular total cholesterol

After seeding in a 6-well culture plate, HepG2 cells were incubated in DMEM containing 10% fetal bovine serum until reaching 70%-80% confluency. After quantifying the cell number, a Cholesterol/Cholesteryl Ester Quantitation Kit (K603-100; Biovision, Inc., Milpitas, CA, United States) was used to extract and measure the intracellular TC according to the manufacturer’s instructions. The intracellular total cholesterol (TC) quantity was expressed as μg/10⁶ cells.

RNA was isolated from HepG2 cells using a Total RNA Kit (R6834; Omega Bio-Tek, Inc., Norcross, GA, United States), and then converted into cDNA by PrimeScript^® RT Reagent Kit (DRR027A; TaKaRa, Shanghai, China). One-fourth of the cDNAs were subjected to qRT-PCR amplification (4367659; Applied Biosystems of Thermo Fisher Scientific). The relative mRNA levels of SREBP2 and HMGCR were measured using the comparative Ct method (∆∆Ct) with β-actin as the internal control. The following primers were used for PCR amplification: SREBP2, sense 5’-CCCTTCAGTGCAACGGTCATTCAC-3’ and antisense 5’-TGCCATTGGCCGTTTGTGTC-3’; HMGCR, sense 5’-CTTGTGTGTCCTT GGTATTAGAGC-3’ and antisense 5’-ATCATCTTGACCCTCTGAGTTACAG-3’; β-actin, sense 5’-CATCCGCAAAGACCTGTACGC-3’ and antisense 5’-AGTACTTGCGCTCAGGAGG AG-3’ (Sangon, Shanghai, China).

Total RNAs including miRNAs were isolated with the mirVana miRNA Isolation Kit (AM1560; Ambion of Thermo Fisher Scientific) according to the manufacturer’s instructions. The high-capacity cDNA reverse transcription kit (4368814; Applied Biosystems) and Bulge-Loop^TM miRNA qRT-PCR Primer Set (MQP-0105; Ribobio, Guangzhou, China) were used to convert selected miRNAs to cDNAs. Expressions of selected miRNAs were analyzed by qRT-PCR using Power SYBR^® Green PCR Master Mix (4367659; Applied Biosystems). RNU6B was used to normalize the mature miRNAs expression levels (MQP-0105; Ribobio).

Cell extracts were isolated in lysis buffer (150 mmol/L NaCl, pH 7.5, 1 mmol/L EDTA, 50 mmol/L Tris-Cl, 10% glycerol, 1% NP-40, and protease inhibitors) for 30 min on ice. The Pierce BCA assay (23225; Pierce Biotechnology, Inc., Rockford, IL, United States) was used to determine the protein concentration. Samples (100 μg) were separated on 10% SDS-polyacrylamide gels and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). The membranes were incubated with anti-β-actin (sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, United States), anti-SREBP2 (ab30682; Abcam, Cambridge, United Kingdom), anti-HMGCR (AP6577c; Abgent, San Diego, CA, United States), or anti-His (sc-803; Santa Cruz Biotechnoogy, Inc.) antibodies in blocking buffer for 2 h at room temperature. After the membranes were incubated with secondary antibodies, the enhanced chemiluminescence system (32209; Thermo Fisher Scientific) was used to detect the immunoreactive bands.

Transfection assays were performed using jetPRIME™ (Polyplus-transfection Inc., Illkirch, France). Either pGL4.10-SREBP2-P (250 ng) or pmirGLO-SREBP2 3’-UTR (250 ng) and the Renilla luciferase vector (pRL-TK; 25 ng) were transfected into HepG2 cells 24 h after seeding in a 48-well culture plate. For some experiments, HepG2 cells were co-transfected with pcDNA3.1/myc-His(-)-core with or without miR-185-5p mimic/inhibitor, whereas the pcDNA3.1/myc-His(-) empty vector and the indicated miR-185-5p negative control were used to normalize the amount of DNA. The cells were harvested and then lysed using the passive lysis buffer (E1941; Promega) at 24 h or 48 h after transfection. A microplate luminometer was used to measure SREBP2 promoter activity and/or SREBP2 3’-UTR activity in some experiments according to the manufacturer’s instructions for the Dual-Luciferase-Reporter Assay System (E1910; Promega). The miR-185-5p mimics/inhibitors and the corresponding negative controls were transfected according to the manufacturer’s protocols (4464014/4464009; Life Technologies).

Data analysis from three independent experiments was performed by the paired Student’s t test. All data are presented as the means ± SE. P < 0.05 was considered statistically significant. The statistical method evaluate by the author Min Li.

To investigate the effects of HCV core protein on lipid metabolism, we cloned the HCV core gene from CHC patients of genotype 1b. Western blotting was performed to confirm whether HCV core protein was expressed or not. The results indicated that HCV core protein was expressed successfully after transient transfection in HepG2 cells (Figure 1A). In HepG2 cells expressing core protein, the intracellular TC was significantly increased (P = 0.001; Figure 1B).

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Figure 1 Hepatitis C virus core protein upregulates expression of sterol regulatory element binding protein 2 and intracellular total cholesterol levels in HepG2 cells. A: HCV genotype 1b core protein was transfected into HepG2 cells and determined by Western blot 48 h after transfection; B: Intracellular TC levels were determined at 72 h after core protein transfection (n = 3, ^bP < 0.01); C: HCV core protein or a control vector was transfected into HepG2 cells. At 24 h after transfection, total RNA was isolated. Then quantitative PCR was performed to determine the relative mRNA levels of SREBP2 and HMGCR. β-actin was used as the internal control (n = 6, ^aP < 0.05, ^bP < 0.01 core vs control); D: HCV core protein or a control vector was transfected to HepG2 cells and SREBP2 and HMGCR expressions were analyzed by Western blot after 48 h; E: The -933 to +263 region of human SREBP2 promoter was amplified using genomic DNA isolated from HepG2 cells as template and cloned into pGL4.10 empty vector, named as SREBP2-P. The luciferase activity of SREBP2-P was determined 24 h after transfection with/without core protein expression (n = 3, ^bP < 0.01 core vs control). HCV: Hepatitis C virus; SREBP2: Sterol regulatory element binding protein 2; TC: Total cholesterol.

Further results suggested that the expression of several lipid-associated genes were upregulated by the HCV core protein, including SREBP1c, SREBP2, HMGCR, HMGCS, and Sirt1 (data not shown). As SREBP2 has been reported to play a key role in cholesterol metabolism, and HMGCR acts not only as the rate-limiting enzyme in cholesterol synthesis, but also a predominant target gene of SREBP2, the effects of the HCV core protein on SREBP2 and HMGCR were investigated. The results showed that core protein increased both the mRNA level (P = 0.009 and 0.037, respectively; Figure 1C) and the protein expression (Figure 1D) of SREBP2 and HMGCR. To explore the mechanism by which HCV core protein upregulates SREBP2 and induces steatosis, the impact of core protein on SREBP2 promoter activity was investigated. The results from the luciferase assay indicate that the SREBP2 promoter activities were enhanced in core-expressing cells (P = 0.004; Figure 1E).

miRNAs have been recently suggested to regulate lipid metabolism genes involved in fatty acid oxidation and cholesterol homeostasis post-transcriptionally[12]. So we proposed that miRNAs may be involved in the regulation of SREBP2 expression by the HCV core protein. To examine whether miRNAs indeed bind to its putative binding sites on the SREBP2 3’-UTR, the pmirGLO-SREBP2 3’-UTR luciferase-reporter construct was transfected into HepG2 cells, and luciferase activity was determined. In pmirGLO-SREBP2 3’-UTR-transfected cells, luciferase activity was repressed significantly compared with the controls, suggesting that certain miRNAs may be involved in the regulation of SREBP2 expression (P < 0.001; Figure 2A). In cells overexpressing core protein, the luciferase activity of SREBP2 3’-UTR was partially restored (P = 0.001). Five miRNAs were predicted to target SREBP2 by a target prediction algorithm (http://www.microrna.org/), including miR-542-3p, 24-3p, 185-5p, 874, and 543. Of these, miR-185-5p (previous ID: miR-185) was also predicted by DIANAmicroT_v3_targets, Targetscan, PITA, and miRDB, to regulate SREBP2 (Table 2). In addition, miR-185-5p is evolutionarily conserved throughout vertebrates[13]. Moreover, miR-185-5p was downregulated by HCV core protein (P = 0.041; Figure 2B), suggesting that misregulation of miR-185-5p by HCV core protein may be an event responsible for SREBP2 overexpression.

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Figure 2 Hepatitis C virus core protein decreases the expression of miR-185-5p. A: The 3’ untranslated region of sterol regulatory element binding protein 2 was co-transfected with control vector (middle) or core protein (right) into HepG2 cells, after 48 h, the relative luciferase activity was detected; Renilla luciferase activity acted as internal control for each transfected well (n = 3, ^bP < 0.01 core vs control); B: 48 h after transfection of a vector expressing the core protein, expression of selected miRNAs was analyzed by quantitative real-time PCR according to the prediction results. RNU6B was used to normalize the mature miRNAs expression levels (n = 3, ^aP < 0.05 core vs control).

miRNAs can pair imperfectly to MREs within 3’-UTRs of target mRNAs to suppress gene expression post-transcriptionally. Bioinformatics analyses revealed that there are at least four putative MREs for miR-185-5p within the SREBP2 3’-UTR. A mutagenesis assay indicated that mutation of the seed sequence binding sites for miR-185-5p in the SREBP2 3’-UTR (SREBP2 3’-UTR-mut a and c) caused a restoration of luciferase activity, even approaching that of the wild-type 3’-UTR (P = 0.033; Figure 3A). Thus, a specific interaction between the SREBP2 3’-UTR and miR-185-5p was confirmed. In contrast, compared with the luciferase activity of wild-type SREBP2 3’-UTR, overexpression of miR-185-5p caused a more drastic repression (P = 0.021; Figure 3B). Finally, we investigated the influence of miR-185-5p on SREBP2 mRNA levels by qRT-PCR in the HepG2 cells. Overexpression of miR-185-5p mimics resulted in significantly reduced SREBP2 mRNA levels (P = 0.022; Figure 3C). Western blot analysis indicated that SREBP2 protein expression was also reduced in miR-185-5p-overexpressing cells (Figure 3D). However, the miR-185-5p inhibitor enhanced SREBP2 protein expression (Figure 3E), indicating that the SREBP2 mRNA and protein expression are regulated by miR-185-5p in HepG2 cells. In the HepG2 cell line, the effect of the miR-185-5p inhibitor was not as strong as the mimic, because the endogenous miR-185-5p expression level was low in this cell line[14]. Overall, miR-185-5p negatively regulated SREBP2 expression as a result of interaction with the SREBP2 mRNA 3’-UTR, which lead to reduced SREBP2 mRNA and protein levels.

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Figure 3 miR-185-5p targets the 3’-untranslated region of sterol regulatory element binding protein 2. A: Luciferase activity was quantified in HepG2 cells transfected with a control luciferase reporter plasmid (Con Luc), SREBP2 3’ UTR-containing reporter plasmid (3’-UTR) and SREBP2 3’ UTR mutant (mut a, b, c, and d) 48 h after transfection (n = 3); B: For overexpression studies, 30 nmol/L miR-185-5p mimic or negative control miRNA was co-transfected with pmirGLO-SREBP2 3’-UTR luciferase constructs. At 48 h after transfection, the relative luciferase activity was detected (n = 3); C: HepG2 cells were transfected with miR-185-5p mimic or negative control miRNA (30 nmol/L) for 48 h, and then the mRNA level of SREBP2 was detected (n = 3); D: HepG2 cells were transfected with miR-185-5p mimic or negative control miRNA (30 nmol/L), and the protein expression of SREBP2 was determined 72 h after transfection; E: HepG2 cells were transfected with miR-185-5p inhibitor or negative control miRNA (30 nmol/L), and the protein expression level of SREBP2 was determined 72 h after transfection. SREBP2: Sterol regulatory element binding protein 2; UTR: Untranslated region.

In addition, the miR-185-5p mimic inhibited the luciferase activity, which was enhanced by HCV core protein (P = 0.024; Figure 4A). Moreover, the HCV core protein upregulated the expression of SREBP2, which was repressed by the miR-185-5p mimic (P = 0.034; Figure 4B), further indicating that miR-185-5p involves the upregulation of SREBP2 by the HCV core protein.

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Figure 4 Upregulation of sterol regulatory element binding protein 2 by Hepatitis C virus core protein involves miR-185-5p. A: 30 nmol/L miR-185-5p mimic or negative control miRNA was co-transfected with pmirGLO-SREBP2 3’-UTR luciferase constructs into HepG2 cells overexpressing core protein for 48 h, and the relative luciferase activity was detected (n = 3); B: In the presence of pmirGLO-SREBP2 3’-UTR, 30 nmol/L of miR-185-5p mimic was co-transfected with either HCV core protein or the indicated null vector into HepG2 cells for 48 h, and the relative luciferase activity was then detected (n = 3). SREBP2: Sterol regulatory element binding protein 2; UTR: Untranslated region.

Liver steatosis is the accumulation of lipid in the liver and is a frequent histologic finding in chronic hepatitis C (CHC). Hepatitis C virus (HCV) infection is a major cause of hepatic steatosis. The HCV core protein is known as an inducer of steatosis. The sterol response element binding proteins (SREBPs) play an important role in regulation of lipid synthesis and cholesterol metabolism. The influence of the HCV core protein on this transcription factor could play an important role in the development of steatosis during HCV infection. However, the pathogenesis of liver steatosis induced by HCV and its implications in the prevention and treatment of CHC remain largely unknown.

In this study, the authors investigated the regulation of expression of host genes by HCV core protein to reveal the molecular pathogenesis of HCV-induced steatosis.

This paper focuses on the intracellular cholesterol regulation by HCV core protein via SREBP2. The regulation relationship between HCV core protein and SERBP2 was established. The microRNA miR-185-5p was confirmed in the regulation of SERBP2 expression, and miR-185-5p was the target for HCV core protein. The authors also established the relationship among the HCV core protein, miR-185-5p, and SREBP2 at transcriptional, post-transcriptional, and translational levels.

HCV core protein controls cholesterol homeostasis through regulating SREBP2 expression, and in this course, miR-185-5p plays an important role. The results suggest that SREBP2 and miR-185-5p may represent a new potential target in the pathogenesis of HCV infection.

Liver steatosis is defined as accumulation of lipid in the liver. Epidemiologic studies revealed that HCV infection is a primary cause of liver steatosis. miRNAs are about 22 nucleotide single-stranded small noncoding RNAs.

In this manuscript, the author analyzed the mechanism that HCV core protein can increase intracellular cholesterol synthesis. HCV can regulate SREBP2 expression via miRNA-185-5p. That is, HCV core decreased miR-185-5p, which was suggested to be responsible for the augmentation of SREBP2 levels. The overall findings are novel and represent a new approach to tackle HCV-induced steatosis