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52364miR-143 Regulates Adipocyte DifferentiationFIG. 3. 2-MOE antisense oligonucleotidesare effective inhibitors ofmiRNA activity in cultured cells. A,expression plasmids used in luciferase assayfor miR-143 activity. B, HeLa cellswere transiently transfected with pRL-CMV Renilla luciferase plasmid (Promega),pGL3-miR-143 sensor, and unlessindicated, pCR3-pri-mir-143. ASOs weretransfected at the indicated doses andevaluated for their ability to interferewith miR-143 inhibition of pGL3-miR-143sensor luciferase expression. Luciferaseactivity was assayed 24 h post-transfectionand for each sample was normalizedto Renilla luciferase activity. C, Northernblotting for miR-143 in differentiatingadipocytes treated with 250 nM ASO targetingmiR-143 at days 7 and 10 afterinduction of differentiation. The blot wasreprobed for snRNA U6 as a loadingcontrol.Drosha processing of pri-miRNA transcripts and subsequentlyreduce mature miR levels. 2Increased Protein Levels of Putative miR-143 Target ERK5 inmiR-143 ASO-treated Adipocytes—There have been several effortsto use bioinformatics techniques to identify target mRNAsregulated by miRNAs (4, 22–26). We wanted to test whetherprotein levels of any predicted miR-143 targets were altered inthe miR-143 ASO-treated adipocytes. However, of the miR-143targets proposed by Lewis et al. (4), only antibodies for ERK5/BMK1 were commercially available. Levels of ERK5 protein innormal adipocytes were compared with adipocytes that hadbeen treated with miR-143 ASO before induction of differentiation.After 7 or 10 days of differentiation, the level of ERK5protein, normalized to G3PDH levels, was 2-fold higher in themiR-143 ASO-treated cells (Fig. 4). The levels of ERK5 proteinwere unchanged in adipocytes treated with an ASO targetingmiR-23b, which also inhibited adipocyte differentiation in thisassay (data not shown). These data suggest that the up-regulationof ERK5 is specific to the miR-143 ASO treatment andnot a general result of inhibition of adipocyte differentiation,consistent with the hypothesis that ERK5 is a miR-143 targetgene.DISCUSSIONIn this study we have screened ASOs targeting miRNAs fortheir ability to modulate adipocyte differentiation in a culturedprimary human adipocyte model system. These data, togetherwith a global analysis of miRNA expression patterns, allowedus to identify several miRNAs involved in adipocyte differentiation.2-O-MOE ASOs were a useful tool for dissecting thebiological importance of miRNAs, and our library of miRNAASOs will be of use in a variety of in vitro and in vivo systemsto rapidly uncover the biological roles of miRNAs. In addition,ASOs targeting miRNA may be valuable as therapeutics in avariety of disease indications.One miRNA identified in the screen, miR-143, was of particularinterest. The observation that miR-143 is up-regulated asadipocytes differentiate, taken together with the inhibition ofadipocyte differentiation by miR-143 ASO, indicates that miR-143 normally promotes adipocyte differentiation. Interestingly,2 B. Lollo, C. Esau, E. Peralta, J. Baroldi, R. Quiambao, R. Jain, andS. M. Freier, manuscript in preparation.FIG. 4.Predicted miR-143 target ERK5/BMK1 is up-regulatedin miR-143 ASO-treated adipocytes A, Western blotting for ERK5/BMK1 in differentiating adipocytes treated with 250 nM ASO targetingmiR-143, at days 7 and 10 after induction of differentiation. The blotwas reprobed for G3PDH as a loading control. B, quantitation of Westernblot.miR-143 levels were previously reported to be decreased incolorectal adenocarcinomas compared with normal tissue (27),and the authors were unable to detect miR-143 expression in avariety of human cancer cell lines. Our own efforts to find acancer cell line expressing miR-143 were also unsuccessful(data not shown). This is consistent with a role for miR-143 inpromoting differentiation and suggests that its down-regulationin cancer cell lines may be a cause or a consequence of theundifferentiated state.Numerous bioinformatics approaches to predicting miRNAtargets have been put forward, but direct verification of aDownloaded from www.jbc.org at University of North Carolina at Chapel Hill, on May 20, 2011
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