Effect of miRNA-10b in regulating cellular steatosis level by targeting PPAR-α expression, a novel mechanism for the pathogenesis of NAFLD

Background and Aim: Accumulating evidence supports the effects of miRNA in lipid metabolism, providing a potential linkage between certain miRNA and non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the miRNA expression pattern in a steatotic L02 cell model and explore the function of certain miRNA target pairs. Methods: The cell model was established by culturing L02 cells with a high concentration of free fatty acid. Micro-array and stem-loop reverse transcription polymerase chain reaction (RT–PCR) were utilized to detect dysregulated miRNA, whereas computational algorithms were used for target prediction. Real time RT–PCR, Western blot, luciferase activity measurement, and other techniques were employed for target verification. Results: Seventeen upregulated and 15 downregulated miRNA were found in steatotic L02 cells, while miRNA-10b was proven to regulate the steatosis level. Peroxisome proliferator-activated receptor-α (PPAR-α) was also found to participate in steatosis, as its protein level was decreased in steatotic L02 cells and its overexpression by transfection into the PPAR-α–pcDNA 3.1 vector could partially alleviate steatosis. We further found that PPAR-α is the direct target of miRNA-10b as it showed significantly changed protein expression, but a relatively unchanged mRNA level in steatotic L02 cells transfected with pre-miRNA-10b and anti-miRNA-10b. Moreover, the action of miRNA-10b on PPAR-α depends on the presence of a single miRNA-10b binding site, as the activity of a luciferase reporter carrying the mutant PPAR-α 3′-untranslated region was not reduced by the expression of miRNA-10b. Conclusion: The established miRNA profile of the steatotic L02 cell model and the novel effect of miRNA-10b in regulating hepatocyte steatosis may provide a new explanation of the pathogenesis of NAFLD.