Effect of miRNA-10b in regulating cellular steatosis level by targeting PPAR-alpha 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-alpha (PPAR-alpha) 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-alpha-pcDNA 3.1 vector could partially alleviate steatosis. We further found that PPAR-alpha 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-alpha depends on the presence of a single miRNA-10b binding site, as the activity of a luciferase reporter carrying the mutant PPAR-alpha 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.