microRNA-425-5p对3T3-L1前体脂肪细胞增殖、分化的影响

为了探究miR-425-5p对小鼠3T3-L1前体脂肪细胞增殖、分化的影响效应,本试验采用实时荧光定量PCR检测miR-425-5p组织和细胞表达水平;运用CCK8、EdU、油红染色、甘油三酯含量分析等分别检测miR-425-5p对前体脂肪细胞增殖、分化的影响;利用生物信息学软件和双荧光素酶报告试验分别预测、验证miR-425-5p调控前体脂肪细胞分化的靶基因。结果表明,miR-425-5p在肥胖小鼠脂肪组织中低表达,在前体脂肪细胞增殖、分化过程中动态表达;与阴性对照相比,过表达miR-425-5p可促进前体脂肪细胞增殖,抑制脂肪细胞分化标志基因(PPARγ、C/EBPα、FAS等)表达,减少脂滴和甘油三酯积累;抑制miR-425-5p表达可抑制前体脂肪细胞增殖,阻止前体脂肪细胞诱导分化。在前体脂肪细胞分化过程中,过表达或抑制miR-425-5p可分别抑制或促进IGF1基因表达;与阴性对照相比,过表达miR-425-5p可抑制IGF1基因3′-UTR荧光活性,而突变miR-425-5p种子序列与IGF1基因3′-UTR的绑定位点可解除该抑制效果。综上所述,miR-425-5p可促进3T3-L1前体脂肪细胞增殖,并可直接靶向IGF1负向调控其分化。

Effect of miR-425-5p on Proliferation and Differentiation of 3T3-L1 Preadipocyte

In order to explore the effect of miR-425-5p on the proliferation and differentiation of 3T3-L1 preadipocyte,the expression of miR-425-5p in tissues and cells were measured by Real-time PCR,the effect of miR-425-5p on the proliferation and differentiation of preadipocyte were measured by CCK8,EdU,oil red O staining and triglyceride detection methods,respectively.The target genes of miR-425-5p regulation preadipocyte differentiation were predicted and identified using bioinformatics softwares and dual luciferase reporter assays,respectively.The results showed that miR-425-5p was expressed at lower levels in adipose tissues of obese mice,and dynamically expressed during preadipocyte proliferation and differentiation.Compared with negative control,miR-425-5p overexpression promoted preadipocyte proliferation,inhibited marker genes related to adipocyte differentiation such as PPARγ,C/EBPα and FAS genes,and reduced lipid droplets and triglyceride accumulation.Inhibition of miR-425-5p expression inhibited the proliferation and prevented the differentiation of preadipocyte.Further analysis found that overexpression or inhibition of miR-425-5p decreased or increased IGF1 gene expression in preadipocyte differentiation,respectively.Compared with negative control,miR-425-5p overexpression inhibited the fluorescence activity of IGF1 gene 3'-UTR,whereas mutation of the miR-425-5p binding site in IGF1 gene 3'-UTR completely abolished this response.This results indicated that miR-425-5p might promote 3T3-L1 preadipocyte proliferation,whereas negatively regulates its differentiation by targeting IGF1.