miRNA作为内源性非编码小RNA,主要在转录后水平调控基因表达.近年来越来越多的证据表明,miRNA通过调控脂肪细胞分化相关的转录因子和信号通路来影响脂肪细胞的分化.为探究miR-26a对小鼠(Mus musculus)3T3-L1前脂肪细胞分化的影响及具体的调节作用,本研究分别用miR-26a的agomir和antagomir对miR-26a进行过表达和敲除.结果显示,miR-26a的表达量在3T3-L1细胞分化过程中逐渐上升.过表达miR-26a极显著增加了脂肪合成相关基因——过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)、脂肪酸合酶(fatty acid synthase,FAS)和脂蛋白脂酶(lipoprotein lipase,LPL)的mRNA水平(P＜0.01),PPARy和FAS的蛋白水平也显著升高(P＜0.05).同时,细胞内的脂质聚集也明显增加.相反,抑制miR-26a的表达量降低了3T3-L1细胞的分化.通过靶基因预测和双荧光素酶报告系统分析,证明miR-26a能直接结合到同源性磷酸酶-张力蛋白(phosphataseand tensin homolog,PTEN) mRNA的3'UTR,并且导致其miRNA和蛋白水平降低(P＜0.05).本研究的结果表明,miR-26a可能通过直接抑制PTEN来正向的调节3T3-L1细胞的脂肪分化,该结果为miRNA成为治疗肥胖相关疾病的药物靶分子提供了一定的理论依据.%As an endogenous small non-coding RNA (ncRNA),microRNAs (miRNAs) regulate gene expression mainly at post-transcriptional level.In recent years,numerous studies have demonstrated that miRNA could have an impact on adipocyte differentiation by modulating the expression of adipogenic transcription factors and signaling molecules.This study showed that the expression of miR-26a gradually rose on day 4 and reached the maximum level on day 8 during 3T3-L1 cell differentiation.In order to investigate the regulatory effects and mechanism of miR-26a on adipogenesis in 3T3-L1 preadipocyte of mouse (Mus musculus),the miR-26a agomir and antagomir were transfected into 3T3-L1 cells to perform miR26a overexpression and knockdown,respectively.The result showed that miR-26a was significantly overexpressed following agomir transfection on day 2 of differentiation,and the elevated miR-26a expression was maintained until the eighth day after differentiation (P＜0.01).In contrast,miR-26a was effectively inhibited on day 2,4 and 8 after induction when the antagomir was transfected into 3T3-L1 cells (P＜0.01).And overexpression of miR-26a significantly accelerated the relative mRNA expression of genes associated with adipogenesis such as peroxisome proliferator-activated receptor gamma (PPARγ),fatty acid synthase (FAS) and lipoprotein lipase (LPL) (P＜0.01,P＜0.05) on day 2,4 and 8,and also increased the protein level of PPARγ and FAS (P＜0.05) on day 8 after differentiation.MiR-26a overexpression also led to a notable increase in lipid accumulation.In contrast,inhibition of miR-26a expression decreased the relative mRNA expression (on day 2,4 and 8) and the protein level (on day 8) of PPARγ and FAS (P＜0.01,P＜0.05) after differentiation.Although there was no significant change in LPL mRNA on day 8 compared to ago-NC group,a significant decrease was detected on day 2 and 4 after differentiation (P＜0.05).Photomicrograph and quantitative analysis of Oil Red O on day 8 of differentiation also revealed that knocking down miR-26a decreased the formation of neutral lipid droplets.Next,to further determine the function of miR-26a,bioinformatics analysis was carried out to predict direct targets of miR-26a,the result showed that the 3' UTR of phosphatase and tensin homolog (PTEN) contained the target sites for miR-26a.By luciferase reporter assay,the firefly luciferase activity was reduced when co-transfection of pmirGLO-PTEN-3' UTR and miR-26a agomirs (P＜0.01),but remained unchanged in other combinations.To better understand the mechanism of miR-26a during adipogenic differentiation in 3T3-L1,the effect of miR-26a on the expression of PTEN had been further validated and the result revealed that overexpression of miR-26a lead to an obvious decrease in both the mRNA (P＜ 0.01) and protein levels (P＜0.05) of PTEN on day 8 of differentiation.In contrast,when the endogenous miR-26a was knocked down with the synthetic miR-26a antagomirs,the PTEN mRNA (P＜0.01) and protein (P＜0.05) levels were increased compared to the ant-NC group.Together,it was concluded that miR-26a positively regulated 3T3-L1 cell differentiation by directly inhibiting PTEN.Therefore,miR-26a and its target genes may play a role in the pathological progression of obesity,which may provide a novel research direction for biological therapy of obesity-related diseases.
展开▼
