摘要目的 探讨microRNA-20a(miR-20a)对心肌糖代谢功能的影响,揭示miR-20a调节胰岛素抵抗和糖尿病等代谢性疾病的机制.方法 以大鼠H9C2心肌细胞为研究对象,建立胰岛素抵抗的细胞模型,应用实时定量逆转录聚合酶链反应(qRT-PCR)检测胰岛素抵抗心肌细胞内差异表达的microRNAs;采用miRanda靶基因预测分析筛选与葡萄糖转运蛋白4(Glut4)基因具有互补结合位点的microRNA;应用细胞转染技术在正常和胰岛素抵抗心肌细胞内分别转染miR-20a类似物与抑制物及相应的阴性对照物,采用qRT-PCR检测各组内miR-20a和Glut4基因表达水平,Western blotting法检测各组内Glut4蛋白表达水平;应用葡萄糖消耗和摄取实验检测各组内葡萄糖代谢水平.采用单因素方差分析及t检验进行数据分析.结果 胰岛素抵抗细胞内miR-20a相对表达量明显高于正常对照组(3.14±0.12比1.00±0.00,t=17.79,P<0.01),Glut4基因和蛋白相对表达明显低于正常对照组(0.58±0.09比1.00±0.00,t=4.71,P<0.05;0.29±0.04比1.00±0.00,t=17.18,P<0.01),miR-20a与人、大鼠、小鼠的Glut43′UTR均存在互补结合位点.转染miR-20a类似物组Glut4基因和蛋白表达显著低于对照组(0.36±0.11比1.00±0.00,t=5.79,P<0.05;0.25±0.05比1.00±0.00,t=13.87,P<0.01);葡萄糖消耗和摄取水平显著低于对照组(48.74±3.95比100.00±0.00,t=12.97,P<0.01;211.30±13.30比350.30±24.55,t=9.77,P<0.05).转染miR-20a抑制物组内Glut4基因和蛋白相对表达较胰岛素抵抗组显著升高(3.63±0.31比1.00±0.00,t=8.39,P<0.01;3.28±0.41比1.00±0.00,t=5.55,P<0.05);葡萄糖消耗和摄取能力显著高于胰岛素抵抗组(240.30±34.12比100.00±0.00,t=3.77,P<0.05;267.70±31.86比122.10±12.94,t=4.33,P<0.05).结论 MicroRNA-20a影响正常和胰岛素抵抗心肌细胞糖代谢水平,可能与其靶向调节Glut4表达有关.

abstractsObjective To investigate the effect of microRNA-20a (miR-20a) on glucose metabolism in H9C2 cardiomyocytes, and reveal the mechanism of miR-20a on regulating insulin resistance and metabolic diseases. Methods The H9C2 cardiomyocytes was taken as the research object to establish the cell model of insulin resistance. The differential expression level of microRNAs was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The miRanda was applied to screen complementary binding sites between microRNA and Glut43′UTR. The H9C2 cells were transfected with miR-20a mimic and negative control (NC), the insulin resistant cells were transfected with miR-20a inhibitor and relative NC, respectively. To apply qRT-PCR for detecting expression levels of miR-20a and Glut4 mRNA. Western blotting was used to detect expression level of Glut4 protein in each group. Glucose consumption and uptake assay were used to measure glucose metabolism level in each group. Data were analyzed with one-way analysis of variance test or t test. Results Compared with control group, the expression of miR-20a was significantly increased in insulin resistance group (3.14 ± 0.12 vs 1.00 ± 0.00, t=17.79, P<0.01), the Glut4 mRNA and protein levels were obviously decreased in insulin resistance group (0.58 ± 0.09 vs 1.00 ± 0.00, t=4.71, P<0.05; 0.29 ± 0.04 vs 1.00 ± 0.00, t=17.18, P<0.01). There were complementary binding sites between miR-20a and Glut43′UTR. Compared with control group, the Glut4 mRNA and protein levels were decreased in miR-20a mimic group (0.36±0.11 vs 1.00±0.00, t=5.79, P<0.05;0.25±0.05 vs 1.00±0.00, t=13.87, P<0.01). Glucose consumption and uptake levels were lower than those of control group respectively (48.74±3.95 vs 100.00±0.00, t=12.97, P<0.01;211.30±13.30 vs 350.30±24.55, t=9.77, P<0.05). The Glut4 mRNA and protein levels were obviously increased in miR-20a inhibitor group compared with insulin resistance group (3.63±0.31 vs 1.00±0.00, t=8.39, P<0.01;3.28±0.41 vs 1.00±0.00, t=5.55, P<0.05). Glucose consumption and uptake levels were increased than those of insulin resistance group respectively (240.30±34.12 vs 100.00±0.00, t=3.77, P<0.05;267.70±31.86 vs 122.10±12.94, t=4.33, P<0.05). Conclusion MicroRNA-20a may affect glucose metabolism level of normal and insulin resistant H9C2 cardiomyocytes by regulating Glut4 expression.