Expression and function of miR-196a-1 during 3T3-L1 adipocyte differentiation | miR-196a-1在3T3-L1脂肪细胞分化中的表达及作用

Chino. 为研究miR-196a-1在脂肪形成中的作用，从3T3-L1细胞基因组中扩增miR-196a-1前体序列，构建miR-196a-1的表达载体，获得稳定表达miR-196a-1的3T3-L1细胞株，成脂诱导后检测脂肪细胞分化关键转录因子的mRNA表达和脂滴累积情况。结果表明：miR-196a-1在3T3-L1细胞分化过程中表达上调，在诱导分化的第2天达到高峰，并在之后的分化过程中恢复到正常水平；稳定表达miR-196a-1的3T3-L1细胞株中miR-196a-1的持续高水平表达导致脂肪形成关键基因过氧化物酶体增殖物活化受体γ(PPARγ)、CCAAT/增强子结合蛋白α(C/EBPα)和脂蛋白脂酶(LPL)的mRNA水平及脂滴累积明显增加。这些结果指明，miR-196a-1促进3T3-L1脂肪细胞分化，利用所构建的稳定表达miR-196a-1的细胞株可进一步研究miR-196a-1调节脂肪形成的分子机制。

Inglés. 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 were 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.