Abstract
Objective: To explore the role and its molecular mechanism of miR-328 during the differentiation of embryonic stem cells (ESCs) into insulin-producing cells (IPCs) in vitro. Method: Mouse embryonic stem cell line-mESCs-Nanog-GFP was induced in conditioned medium and divided into negative control group, miR-328 agomir transfected group, miR-328 antagomir transfected group and transforming growth factor β2 (TGF-β2) siRNA transfected group. The function of IPCs was identified by real-time quantitative PCR (qPCR) detecting system and immunofluorescence in above-mentioned groups. Methods of qPCR, immunofluorescence and enzyme-linked immunosorbent assay (ELISA) were used to detect effects of overexpression and inhibition of miR-328 on differentiation of multilineage precursor cells. We predicted the binding sites of miR-328 and TGF-β2 by performing the bioinformatics analysis. Dual luciferase reporter gene and Western blotting were employed to identify the regulatory relationship between miR-328 and TGF-β2. Results: mESCs could be transfected with miR-328 agomir, with an efficacy of 70%-80%. Up-regulated miR-328 in MPCs reduced the RNA expression of several key transcription factors which were crucial for early pancreatic development. Additionally, the insulin released by IPCs decreased in response to glucose stimulation (all P<0.05). However, overexpression of miR-328 led to the decrease of protein level of insulin and Nkx6.1 (all P<0.05). Transfection of miR-328 antagomir had the opposite effects (P<0.05). The dual luciferase reporter gene assay revealed that miR-328 functioned via binding to the 3' non-coding region (3'-UTR) of the TGF-β2. Western blotting indicated that miR-328 regulated protein expression. After knockdown of miR-328, the relative expression of TGF-β2 was 1.00±0.01. After co-transfection of miR-328 antagomir and TGF-β2 siRNA, the relative expression of TGF-β2 was 0.80±0.03. After downregulating TGF-β2, the relative expression of TGF-β2 was 0.20±0.01. Knockdown of TGF-β2 down-regulated the expression of early pancreatic transcription factors (P<0.05) and inhibited Pdx1(+)cell differentiation. Conclusion: miR-328 can inhibit the differentiation of ESCs into IPCs via binding to 3' UTR of TGF-β2, and provide a new regulatory pathway for the treatment of diabetes with stem cells.
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