目的 探讨miR-29a调控心肌肥大的分子机制.方法 利用荧光素酶报告基因实验验证miR-29a与其潜在靶基因PPARδ/β的靶向关系.体外分离培养新生SD大鼠心肌细胞,分别以5 mmol/L葡萄糖(正常组)、25 mmol/L葡萄糖(高糖诱导组)和转染miR-29a抑制剂+25 mmol/L葡萄糖(实验组)培养48 h,应用Image J软件测量单细胞表面积,用qRT-PCR法检测各组miR-29a以及心肌肥大标志基因β-MHC和BNP mRNA的表达,采用Western blot法检测各组心肌细胞PPARδ/β蛋白表达情况.结果 荧光素酶报告基因实验显示调控能量代谢的基因PPARδ/β是miR-29a的潜在靶点.高糖诱导组单细胞表面积,β-MHC、BNP mRNA表达水平和miR-29a表达水平均明显高于正常组(P均＜0.05),而PPARδ/β蛋白表达水平显著低于正常组(P＜0.05);实验组单细胞表面积,β-MHC、BNP mRNA表达水平和miR-29a表达水平均明显低于高糖诱导组(P均＜0.05),PPARδ/β3蛋白表达水平显著高于高糖诱导组(P＜0.05),各指标与正常组比较差异均无统计学意义(P均＞0.05).结论 miR-29a通过影响心肌细胞的能量代谢而调控心肌肥大.%Objective It is to find the molecular mechanism of cardiac hypertrophy regulated by miR-29a.Methods The Luciferase Reporter Gene Assays were used to verify whether miR-29a can target the potential gene PPARδ/β.Neonatal SD rat cardiomyocytes were cultured in vitro.They were divided into three groups and received different treatment as followings for 48 hours:5 mmol/L glucose (normal group),25 mmol/L glucose (high glucose induced group),transfected with miR-29a inhibitors and 25 mmol/L glucose (experimental group).Image J software was used to measure the cell surface areas.Q-PCR was used to test miR-29a expression levels and the mRNA expression levels of β-MHC and BNP,which are marker genes of cardiac hypertrophy.Three groups protein levels of PPARδ/β were confirmed by Western blotting.Results The Luciferase Reporter Gene Assays showed PPARδ/β gene was targeted by miR-29a.The single cell surface areas,miR-29a expression and β-MHC and BNP mRNA expression levels of high glucose induced group were significantly increased,but the protein levels of PPARδ/ β were significantly decreased,compared to normal group (P ＜ 0.05).Moreover,these effects induced by high glucose can be reversed and recovered to normal in experiment group (P ＜ 0.05).Conclusion miR-29a could regulate cardiac hypertrophy by controlling energy metabolism of cardiomyocytes.
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