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


CONCLUSIONS
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.
Department of Infectious disease, First Affiliated Hospital, Medical School, Zhejiang University, Zhejiang, China.
Lin
L
Zheng
Guo-cai
GC
Lv
Jifang
J
Sheng
Yi-da
YD
Yang
D020413:3' Untranslated Regions; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D019295:Computational Biology; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D005234:Fatty Liver / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020869:Gene Expression Profiling / Q000379:methods; D017930:Genes, Reporter; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D050356:Lipid Metabolism / Q000235:genetics*; D035683:MicroRNAs / Q000378:metabolism*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D047493:PPAR alpha / Q000235:genetics* / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014280:Triglycerides / Q000378:metabolism
D020413:3' Untranslated Regions; D005230:Fatty Acids, Nonesterified; C547183:MIRN10 microRNA, human; D035683:MicroRNAs; D047493:PPAR alpha; D012333:RNA, Messenger; D014280:Triglycerides