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.

Keywords: Case-Control Studies, Biopsy, Gene Expression, Homeodomain Proteins - metabolism, Non-alcoholic Fatty Liver Disease - pathology, RNA, Messenger - metabolism, Sirtuin 1 - metabolism, Transcription Factors - metabolism, Triglycerides