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Abstract To evaluate the influence of miR-196a-1 on 3T3-L1 adipocyte differentiation, the precursor of miR-196a-1 was amplified from the 3T3-L1 cells genomic DNA, and then miR-196a-1 expression vector was constructed, and the miR-196a stable expressing cells were obtained. The transfection of pcDNA3.0-miR-196а-1 into 3T3-L1 cells was followed by the positive clones selection of G418 and the miR-196a stable expressing cells were obtained. The miR-196a stable expressing cells were treated with an adipogenic stimulus, and the expression of adipogenic marker genes and the lipid accumulation were detected. The results showed that miR-196a-1 was up-regulated during adipocyte differentiation. The target sequence which was inserted into pcDNA3.0 was about 340 bp, and the sequencing result was consistent with miR-196a-1. The positive clones could express miR-196a-1 stably, and the expression level of miR-196a-1 increased nearly 10-fold compared to control cells. In the miR-196a-1 stable expressing cells, the mRNA levels of adipogenic marker genes: peroxisome proliferator-activated receptor γ(PPARγ), CCAAT enhancer binding protein α(C/EBPα) and lipoprotein lipase(LPL) and the lipid accumulation increased markedly after adipogenic induction. These results suggest that miR-196а-1 was involved in adipogenesis. The miR-196a-1 stable expressing cell line can be used for further studies of the function and mechanism of miR-196a-1 in the adipogenesis.
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Received: 23 March 2010
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