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.
目的： 探讨miR-328在小鼠胚胎干细胞体外分化胰岛素分泌细胞过程中的调控作用及分子机制。 方法： 小鼠胚胎干细胞系mESCs-Nanog-GFP经条件培养基诱导后，分为阴性对照组、转染miR-328激动剂组、转染miR-328拮抗剂组和转染转化生长因子β2（TGF-β2）小干扰RNA组。通过实时荧光定量PCR（qPCR）、免疫荧光鉴定胰岛素分泌细胞功能；qPCR、免疫荧光及酶联免疫吸附（ELISA）检测多系前体细胞过表达及抑制miR-328对胰岛素分泌细胞分化的影响；生信分析预测miR-328与TGF-β2结合位点，双荧光素酶报告基因及Western印迹法验证两者调控关系。 结果： miR-328激动剂可成功转染小鼠胚胎干细胞，转染效率为70%~80%，使胰岛功能相关基因表达均有不同程度下降，且葡萄糖刺激胰岛素释放量减少，胰岛素、NK6同源盒1蛋白表达下降（均P<0.05）；转染miR-328拮抗剂上调胰岛功能相关基因及转录因子表达（均P<0.05）。双荧光素酶报告基因实验发现miR-328可结合至TGF-β2的3′端非编码区（3′-UTR），Western印迹检测显示转染miR-328拮抗剂组TGF-β2蛋白相对表达量1.00±0.01，共转染miR-328拮抗剂组和TGF-β2小干扰RNA组蛋白相对表达量0.80±0.03，转染TGF-β2小干扰RNA组蛋白相对表达量0.20±0.01。敲降TGF-β2下调胰腺早期转录因子表达（P<0.05），抑制Pdx1(+)细胞分化。 结论： miR-328能够靶向TGF-β2抑制小鼠胚胎干细胞向胰岛素分泌细胞的分化，为糖尿病干细胞治疗提供了新的调控途径。.
Keywords: Cell differentiation; Embryonic stem cells; Insulin-secreting cells; MicroRNAs.