SGBS adipocytes overexpressing miR-192* or a non-targeting control miRNA

To determine whether expression microRNAs (miRNAs) in visceral adipose tissue (VAT) is altered in human non-alcoholic fatty liver disease (NAFLD), we first performed miRNA profiling of VAT from subjects with (n=15, macroscopic liver fat >30%) or without (<5%, non-NAFLD, n=15) NAFLD. VAT miRNA expression profiles in the two groups were similar except for a single miRNA, miR-192*, which was 1.9-fold increased in NAFLD. We next overexpressed miR-192* in cultured human adipocytes, measured its effect on the cellular triglyceride content, and characterized these adipocytes by transcriptomic profiling followed by pathway analysis, qPCR and quantification of select cellular proteins. SGBS preadipocytes were transduced with shRNA miR-192* or a non-targeting control miRNA. Growth media was added after four hrs of incubation. The cells were grown in standard medium for 48 hrs without selection, after which the growth medium was replaced with a medium containing 0.5 μg/ml puromycin (Sigma-Aldrich). SGBS adipocytes overexpressing miR-192* or a non-targeting control miRNA were lysed and total RNA was isolated and purified. After verification of the quality of RNA, we performed gene expression analysis in a modified standard Agilent microarray (023043) containing 205 free positions (Agilent) to was verified by 2100 Bioanalyzer (Agilent).