Abstract：Abstract 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 miR-26a 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.
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