HepG2 cells were cultured in DMEM containing 10% foetal bovine serum, 1% of an antibiotic cocktail containing penicillin (10000 U/mL) and streptomycin (10000 μg/mL), 1% non-essential amino acid solution, 1% L-glutamine and 5.0 mmol/L glucose, in a humidified environment with 5% CO₂ at 37 °C. MiR-185, control microRNA, anti-miR-185 and control antisense oligonucleotides (ASOs) were synthesized by RiboBio Co., Ltd. (Guangzhou, China); the oligonucleotides were transfected into HepG2 cells using the Lipofectin transfection reagent (Invitrogen, United States), according to the manufacturer’s instructions.

The animal study was approved by the ethics committee of Harbin Medical University (Harbin, Heilongjiang, China) and experiments were conducted according to the National Institutes of Health guidelines for humane treatment of laboratory animals. Eight-week-old male C57Bl/6 mice were obtained from Harbin Medical University Laboratories (Harbin, Heilongjiang, China) and housed with a 12 h light/dark cycle, allowing free access to water and pellet chow. To assess miR-185 expression levels, C57BL/6 mice were divided into two groups, fed a normal laboratory diet and an HFD (30% fat, 15% protein, 45% carbohydrate, and 1.15% cholesterol), respectively, for 12 wk. At 4, 8 and 12 wk, liver tissues were harvested for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) experiments respectively.

For miR-185 treatment, 20 eight-week-old male C57BL/6 mice were fed an HFD for 12 wk. Then, half of the mice (n = 10) were intravenously injected with 20 mg/kg miR-185 while the other half, used as controls (n = 10), were administered equal amounts of the control microRNA weekly for 8 wk. Mouse body weights were recorded weekly.

To determine whether miR-185 acts as a regulator in lipid metabolism, we first quantitated alterations in miR-185 expression in response to palmitic acid (PA) stimulation. The HepG2 cells were treated with 1.0 × 10^-4 or 5.0 × 10^-4 mmol/L PA, and qRT-PCR was used to assess miR-185 levels after 24 h and 48 h of culture.

Total RNA was isolated from the liver tissue or cultured cells using a High Pure miRNA Isolation Kit (5080576001, ROCHE, Germany) according to the manufacturer’s instructions. Then cDNA was synthesized using a miRcute miRNA cDNA kit (KR201-01, Invitrogen, United States). qRT-PCR was performed on an Applied Biosystems 7900HT Sequence Detection System (Applied Biosystems, United States).

To assess mRNA levels of SREBP1, SREBP2, fatty acid synthase (FAS), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), total RNA was extracted and purified using Trizol (Invitrogen, United States) and the RNeasy Mini kit (Qiagen), respectively, according to the manufacturers’ instructions. The purified RNA was treated with DNase I and reverse transcribed using the MMLV Reverse Transcriptase First Strand cDNA Synthesis Kit (Invitrogen, United States). All reactions were performed in triplicate, and RNU6-2 or β-actin was selected as an internal control for normalization. Relative fold changes in gene expression were determined by the 2^−ΔΔCt method[14]. The primer sequences used for RT-PCR are listed in Table 1.

HepG2 cells were cultured in 12-well plates and transfected with miR-185 (20 nmol/L), anti–miR-185 (40 nmol/L), or negative controls (con-miR and con-anti-miR). After 48 h, the fatty acid oxidation rate was evaluated by quantifying the oxidation of [1-¹⁴C] oleate into ¹⁴CO₂, as previously described (Yu et al, 1997). The sterol synthesis rate was estimated by measuring the amounts of [¹⁴C] acetate incorporated into cellular sterols, as described previously (Ettinger et al, 1994). Each experiment was performed in triplicate.

Forty-eight hours after transfection, cells were washed in phosphate-buffered saline (PBS) and lysed with 300 μL RIPA lysis buffer (Solarbio, Beijing, China) containing a protease inhibitor mixture (Roche Applied Science, Germany). Proteins were resolved by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dry milk and probed with primary antibodies raised against insulin receptor substrate (IRS) 1 (Abcam, United States), IRS-2 (Santa Cruz, United States), AKT2 (Santa Cruz, United States), PI3K (Santa Cruz, United States), and β-actin (Sigma, United States). After three washes with TBS-T, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, United States). The signals were visualised with an ECL kit (GE Healthcare, CT, United States).

Plasma concentrations of total cholesterol (CHOL), triglycerides (TG), and alanine aminotransferase (ALT) were determined after the last injection using spectrophotometric assay kits (Sigma-Aldrich, St. Louis, MO, United States) according to the manufacturer’s instructions.

Mice were submitted to 8 h fasting and administered insulin by intraperitoneal injection. Then, plasma insulin levels were measured by ELISA and blood glucose levels (in tail vein blood) were assessed at 0, 15, 30, 60, and 120 min after insulin (1.0 U/kg body weight) treatment using a blood glucose meter (One Touch Ultra, Lifescan, United States) according to the manufacturer’s instructions.

To evaluate the effect of miR-185 on hepatic steatosis, liver tissues were harvested from mice after the last injection of miR-185, fixed in 10% formalin solution, and embedded in paraffin. The sections were stained with hematoxylin and eosin according to standard protocols.

Statistical analyses were carried out using SPSS statistical software version 12.0 (SPSS Inc., Chicago, IL, United States). Data are expressed as mean ± SD. Student’s t-test and one way analysis of variance (ANOVA) were used to compare the differences between two or among more than two groups, respectively. P < 0.05 and P < 0.01 were considered statistically significant and highly significant, respectively.

In the presence of palmitic acid, miR-185 expression levels decreased by approximately 20% and approximately 35% after treatment with 1.0 × 10^-4 mmol/L PA (P < 0 .01) and 5.0 × 10^-4 mmol/L PA (P < 0.05), respectively, after 24-h incubation compared with control cells (Figure 1A). This effect was increased after 48-h incubation, with miR-185 expression decreasing by approximately 42% (1.0 × 10^-4 mmol/L PA, P < 0.01) and 60% (5.0 × 10^-4 mmol/L PA, P < 0.05) compared with the control group (Figure 1B). These data suggested that miR-185 expression levels decreased in a time- and dose-dependent manner in response to PA in HepG2 cells.

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Figure 1 MicroRNA-185 expression levels in palmitic acid-treated HepG2 cells. HepG2 cells were treated with 1.0 × 10^-4 or 5.0 × 10^-4 mmol/L PA; qRT-PCR detected microRNA-185 (miR-185) levels after 24-h (A) or 48-h (B) incubation. RNU6-2 was used as an internal control for miR-185. Data are mean ± SEM from three separate experiments. ^aP < 0.05, ^bP < 0.01 vs the normal control group. PA: Palmitic acid; qRT-PCR: Quantitative reverse transcription-polymerase chain reaction.

To evaluate the modulatory effect of miR-185 on lipid metabolism, HepG2 cells were transfected with pre-miR-185 or anti-miR-185, and fatty acid oxidation and sterol synthesis rates were determined. Compared with control cells, HepG2 cells overexpressing miR-185 displayed a stark decrease in the sterol synthesis rate of approximately 30% (P < 0.01; Figure 2A) and an increased fatty acid oxidation rate (1.9-fold, P < 0.01; Figure 2B). Inhibition of miR-185 by anti-miR-185 resulted in a 2.2-fold increase in the sterol synthesis rate (P < 0.01, Figure 2C) and approximately 38% decrease in the fatty acid oxidation rates (P < 0.01, Figure 2D) compared with controls.

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Figure 2 Effect of miR-185 on lipid metabolism in HepG2 cells. HepG2 cells were transfected with pre-miR-185 or anti-miR-185 (an antisense oligonucleotide against miR-185), and fatty acid oxidation and sterol synthesis rates were determined. A: The sterol synthesis rate was determined by the amount of [¹⁴C] acetate incorporated into HepG2 cell sterols after transfection with pre-miR-185; B: The fatty acid oxidation rate was measured by the oxidation of [1-¹⁴C] oleate into ¹⁴CO₂ after transfection with pre-miR-185; C: Sterol synthesis rates in HepG2 cells after transfection with miR-185 inhibitors; D: Fatty acid oxidation rates in HepG2 cells after transfection with miR-185 inhibitors; E: Quantitative reverse transcription-PCR was used to assess the mRNA levels of key lipid metabolism-associated genes in HepG2 cells after overexpression of miR-185; F: mRNA levels of lipid metabolism-associated genes after miR-185 inhibition. Data are mean ± SEM from three separate experiments. ^aP < 0.05, ^bP < 0.01 vs the normal control group. miR-185: microRNA-185; ASO: Antisense oligonucleotide; SREBP-1C: Sterol-regulatory element-binding proteins 1C; SREBP-2: Sterol-regulatory element-binding proteins 2; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase.

Since multiple studies have shown that FAS, HMGCR, SREBP2, and SREBP1c are strongly associated with lipid metabolism, the effect of miR-185 on these lipid metabolism-associated genes was assessed. Gene expression levels of FAS, HMGCR, SREBP2, and SREBP1c were measured after induction or inhibition of miR-185. qRT-PCR data revealed dramatically reduced expression of FAS, HMGCR, SREBP2, and SREBP1c after miR-185 overexpression in HepG2 cells (Figure 2E). Conversely, inhibition of miR-185 in HepG2 cells resulted in increased FAS, HMGCR, SREBP2, and SREBP1c mRNA levels (Figure 2F). These findings indicated that miR-185 regulates fatty acid metabolism and cholesterol homeostasis by suppressing the expression of lipogenic genes in hepatocytes.

The in vitro data described above prompted us to further investigate the role of miR-185 in regulating lipid metabolism in vivo. C57BL/6J mice were fed a normal chow or a high fat diet for 12 wk, and liver tissues were harvested after sacrifice at weeks 4, 8, and 12 for qRT-PCR experiments. MiR-185 expression was decreased by approximately 22% at 8 wk (P < 0.01) and further declined (approximately 30%) at 12 wk (P < 0.01) compared with normal controls. In agreement with the in vitro data, miR-185 expression declined in the HFD group in a time-dependent manner (Figure 3).

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Figure 3 MicroRNA-185 expression levels in C57BL/6J mice fed a high fat diet. C57BL/6J mice were fed a normal chow or a high fat diet for 12 wk and liver tissues were harvested at 4, 8, and 12 wk. The miR-185 levels were determined by quantitative reverse transcription-PCR (n = 10). Data are mean ± SEM from three separate experiments. ^aP < 0.05 vs the normal control group. miR-185: microRNA-185.

To elucidate the regulatory effect of miR-185 on insulin sensitivity and lipid metabolism, c57BL/6 mice were fed a high-fat diet for 12 wk, then treated intraperitoneally weekly with miR-185 (20 mg/kg) for 8 wk. To determine insulin sensitivity, an ITT was performed after the last miR-185 administration. The results showed significantly lower plasma glucose concentrations in the HFD + miR-185 group compared with the HFD + con-miR group at all time points after injection of 1U/kg insulin (Figure 4A).

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Figure 4 Overexpression of microRNA-185 improves insulin sensitivity and reduces liver steatosis. Eight-week-old male C57BL/6 mice were fed a high-fat diet for 12 wk. One half of the mice (n = 10) were injected with miR-185 (20 mg/kg body weight) and the control group (n = 10) received injections of control microRNA for 8 wk. A: ITT was performed after the last injection, and mice were submitted to 8 h fasting before intraperitoneal administration of insulin; B: Body weights were recorded weekly during the treatment period; C-E: Triglycerides, cholesterol and alanine aminotransferase levels were assessed after the last injection; F: At the end of the treatment, liver sections were stained by haematoxylin and eosin stain to assess lipid accumulation (magnification × 400). Where applicable, data are mean ± SEM from three separate experiments, n = 10, ^aP < 0.05 vs the normal control group. HFD: High-fat diet.

The effect of miR-185 overexpression on NAFLD was also assessed. After the 8-wk treatment regimen, no significant difference was observed between the HFD + con-miR and HFD + miR-185 groups in terms of mouse body weights (Figure 4B); TG, CHOL and ALT levels decreased after miR-185 treatment compared with the HFD + con-miR group (Figure 4C-E). In addition, HE staining showed enlarged cells with hepatocellular ballooning in the HFD + con-miR group. Finally, lipid accumulation decreased significantly after miR-185 treatment (Figure 4F). These data suggested that miR-185 overexpression alleviates liver fat content in C57BL/6 mice.

To explore whether miR-185 regulates the insulin signalling pathway, we assessed the expression of key pathway components (IRS-1, IRS-2, PI3K and Akt2) and the phosphorylation of Akt2 and PI3K after transfection of HepG2 cells with pre-miR-185. As shown in Figure 5A, IRS-2 expression was significantly elevated (3-fold) after miR-185 overexpression, whereas the expression levels of IRS-1, PI3K and Akt2 were unchanged. Moreover, both pPI3K and pAkt2 were markedly increased (Figure 5B), suggesting that miR-185 promotes the PI3K/Akt2 pathway by inducing IRS-2 expression rather than IRS-1.

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Figure 5 MicroRNA-185 regulates the insulin signalling pathway in HepG2 cells. A: The HepG2 cells were transfected with pre-miR-185 or pre-miR control and, after 48 h, the protein expression levels of insulin receptor substrate (IRS)-1, IRS-2, phosphatidylinositide 3-kinase (PI3K) and Akt2 were detected by Western blot; B: Effect of miR-185 on Akt2 and PI3K phosphorylation. miR-185: microRNA-185.

Lipid metabolism and the insulin signalling pathway are frequently closely associated with non-alcoholic fatty liver disease (NAFLD). Therefore, identification of the regulators that control lipid metabolism and insulin resistance is critical.

No previous studies have reported that an microRNA co-regulated lipid metabolism and the insulin signalling pathway.

This is the first study to demonstrate that microRNA-185 (miR-185) regulates lipid metabolism and insulin signalling in vivo and in vitro.

Our findings define additional functional relevance of miR-185 that might be useful in the design of therapeutic strategies for treating NAFLD and improving insulin resistance.

Insulin signalling pathways includes the phosphatidylinositide 3-kinase/Akt pathway and Ras/MAPK pathway. MicroRNAs are endogenous non-protein coding small RNA molecules (22 nucleotides in length) that negatively regulate target gene expression by suppressing the translation of specific mRNAs.

This is a well-written paper with well thought out experiments, and each finding in this study may be worth reporting. The paper reports the significance of miR185 in the regulation of lipid metabolism gene expression and blockade of the insulin signalling pathway in vitro and in vivo.