The Impact Of MiR-196b-5p On Adipocyte Differentiation

Background and objective: With the quality of life improved, the incidence of obesity increased year by year, which can lead to insulin resistance, diabetes, cancer, osteoporosis, etc. Thus it is necessary to find more effective strategies to prevent and treat obesity. Since the discovery of mi RNA, a growing number of studies have shown that mi RNA has a certain effect on the differentiation of adipocytes. This study was to investigate the role of mi R-196b-5p in the differentiation of adipocytes, identify the target genes and to elucidate the mechanism by which mi R-196b-5p regulates adipocyte differentiation.Methods: 1. Bone marrow stromal cells of mice were isolated, cultured and induced with adipogenic agents for 1 day. The expression level of mi R-196b-5p was examined by using RT-q PCR. Stromal ST2 cells were transfected with mi R-196b-5p mimics or mi R-196b-5p inhibitor followed by adipogenic treatment. Western blotting, Oil-red O staining and q RT-PCR were conducted to detect the differentiation of adipocytes. 2. The potential target genes of mi R-196b-5p were predicted using online tools. Then the 3 ’UTR fragments of the target genes containing the base-pairing sequences with mi R-196b-5p were PCR-amplified and cloned into luciferase reporter vector. The constructs were cotransfected with either mi R-196b-5p mimics or its negative control to AD-293 cells. Dual luciferase assay was done to study if the potential genes are really the targets of mi R-196b-5p.Results: 1. RT-q PCR showed that the expression of mi R-196b-5p in ST2 cells was increased after adipogenic treatment. This suggests that mi R-196b-5p may be involved in the regulation of adipocyte differentiation. 2. After transfection of mi R-196b-5p mimics, the expression levels of PPARγ, C/EBPα, adipsin and a P2 were up-regulated, and the differentiation of cells was enhanced. By contrast, the transfection of mi R-196b-5p inhibitor led to opposite results. This suggests that mi R-196b-5p promots the differentiation of adipocytes. 3. Tgfbr1, Nr2c2, Rbpj, Foxo1 and Sema3 a were predicted to be potential target genes. Dual luciferase reporter gene assay showed that overexpression of mi R-196b-5p significantly decreased the luciferase activity of 3’UTR constructs of Tgfbr1 and Foxo1, confirming their identity as the real targets of mi R-196b-5p.Conclusion: 1.mi R-196b-5p is significantly up-regulated in the mouse ST2 cells after adipogenic treatment. 2. mi R-196b-5p promotes the differentiation of bone marrow stromal cells into adipocytes. 3. Tgfbr1 and Foxo1 are likely to be targets of the mi R-196b-5p. mi R-196b-5p may promote adipocyte differentiation through targeting Tgfbr1 and Foxo1.