Altered microRNA-9 Expression Level is Directly Correlated with Pathogenesis of Nonalcoholic Fatty Liver Disease by Targeting Onecut2 and SIRT1

Abstract

BACKGROUND MicroRNA-9 (miR-9) was detected in nonalcoholic fatty liver disease (NAFLD) patients to understand the role of miR-9 in NAFLD development. MATERIAL AND METHODS Between February 2014 and February 2015, 105 cases of NAFLD were recruited and confirmed by liver biopsy pathology, including patients with mild NAFLD (n=58) and moderate-severe NAFLD (n=47); nonalcoholic steatohepatitis (NASH) (n=53) and non-NASH (n=52); and 50 healthy participants were regarded as the healthy control group. MiR-9 expression was measured by qRT-PCR. For in vitro experiments, L-02 normal liver cells were divided into normal control group (cultured with original culture medium), dimethyl sulfoxide (DMSO) group (cultured with DMSO) and oleic acid group (cultured with oleic acid to induce fatty change), and MTT assay was used to measure the effect of different oleic acid concentrations on cell proliferation. Nile red staining was used to detect intracellular accumulation of lipid droplets. Further, synthetic miR-9 mimic and its control and miR-9 inhibitors and its control were independently transfected into L-02 cells. RESULTS MiR-9 levels in the mild NAFLD group and moderate-severe NAFLD group were significantly higher than in the healthy control group (both P<0.05). Mean fluorescence intensity of lipid droplets increased with the duration of induction, and were dramatically higher in oleate-treated L-02 cells; intracellular triglyceride (TG) content was also higher. miR-9 levels significantly increased following oleate induction. Importantly, miR-9 levels were significantly elevated upon miR-9 mimic transfection. Conversely, miR-9 level was lowered with miR-9 inhibitors transfection. Additionally, Onecut2 and SIRT1 were identified as miR-9 targets. CONCLUSIONS A positive relationship between miR-9 and steatosis was established with our results that miR-9 mimic transfection decreased intracellular lipid content. Finally, we identified 2 miR-9 targets, Onecut2 and SIRT1, which may be crucial players in NAFLD development.

Figure 1

Comparison of cell morphology and growth, observed by laser scanning confocal microscopy, of normal L-02 cells and steatotic hepatocytes induced by oleic acid, (A) normal L-02 cells (×10); (B) steatotic hepatocytes (×10).

Figure 2

Comparison of cell absorbance (OD) by MTT assay in oleic acid groups with different concentrations of oleic acid (5 g/ml, 10 g/ml, 20 g/ml and 40 g/ml) at 24 h, 48 h and 72 h, respectively, (A) Steatotic hepatocytes induced by oleic acid; (B) Steatotic hepatocytes treated with dimethyl sulfoxide (DMSO).

Figure 3

Fluorescence-based detection of intracellular lipid droplets by Nile red with laser scanning confocal microscopy (10×40), (A) L-02 cells of normal control group before induction; (B) Steatotic hepatocytes observed at 24 h after induction; (C) Steatotic hepatocytes observed at 48 h after induction; (D) Steatotic hepatocytes observed at 72 h after induction; (E) Changes in lipid droplets in cells. * Compared to the normal control (NC) group, P<0.05; ^# Compared to steatotic hepatocytes induced by oleic acid at 24 h, P<0.05; ^@ Compared to steatotic hepatocytes induced by oleic acid at 48 h, P<0.05.

Figure 4

The standard curve of triglyceride (TG), revealing the increased intracellular TG content with the longer duration of oleate induction, which was significantly higher than in the normal control group at 24 h, 48 h, and 72 h. (A) Standard curve of TG; (B) Concentration of TG within the steatotic hepatocytes induced by oleic acid at 24 h, 48 h, and 72 h. * Compared to normal control (NC) group, P<0.05.

Figure 5

Comparison of microRNA-9 expression level between normal L-02 cells and steatotic hepatocytes induced by oleic acid: Changes in microRNA-9 expression after transfection, * Compared to the normal control (NC) group, P<0.05; ^# Compared to non-transfected steatotic cell group, P<0.05; ^@ Compared to the miR-9 mimic group, P<0.05.

Figure 6

The distribution of intracellular lipid droplets in each transfection group observed by laser scanning confocal microscopy (10×40). (A) Normal control (NC) group; (B) Non-transfected steatotic cell group; (C) miR-9 mimic negative control (NC) group; (D) miR-9 inhibitor negative control (NC) group; (E) miR-9 mimic group; (F) miR-9 inhibitor group. (G) The mean fluorescence intensity of lipid droplets in different transfected cells. * Compared to the normal control (NC) group, P<0.05; ^# Compared to the non-transfected steatotic cell group, P<0.05; ^@ Compared to the miR-9 mimic group, P<0.05.

Figure 7

Prediction and identification of miR-9 target genes. (A–D) miR-9 target genes expression detection using RT-PCR (A: Onecut2; B: SIRT1; C: REST; D: CoREST). * Compared to the normal control (NC) group, P<0.05; ^# Compared to the non-transfected steatotic cell group, P<0.05; ^@ Compared to the miR-9 mimic group, P<0.05. (E) Western blotting to assess the expression of Onecut2, SIRT1, REST, and CoREST in the NC group, non-transfected steatotic cell group, mimic NC group, inhibitor NC group, miR-9 mimic group, and miR-9 inhibitors group.

Figure 8

(A) Prediction of biological information and the targeting relationships between SIRT1 and Onecut and miR-9 validated by dual luciferase reporter gene assay. microRNA.org predicts that Onecut2, SIRT1, REST, and CoREST are the target genes of miR-9; (B) Dual luciferase reporter gene results assay confirmed that Onecut2 and SIRT1 are target genes of miR-9 in L-02 cells (* Comparison between miR-9 mimic group and miR-9 mimic negative control (NC) group, P<0.05).