miR-30a在同型半胱氨酸致足细胞凋亡中的作用

Effect of miR-30a on homocysteine-induced podocytes apoptosis

目的探讨miR-30a在同型半胱氨酸(homocysteine,Hcy)诱导足细胞凋亡中的作用。方法体外培养小鼠肾小球足细胞,分为Control组(0μmol/L Hcy)与Hcy组(80μmol/L Hcy),干预细胞48 h后,流式细胞术检测足细胞凋亡水平;实时荧光定量PCR(qRT-PCR)检测miR-30a的表达水平;巢式甲基化特异性PCR(nMS-PCR)检测miR-30a启动子区DNA甲基化水平;透射电镜观察感染miR-30a前体(miR-30a mimic)和抑制物(miR-30a inhibitor)后足细胞凋亡及超微结构的变化。结果与Control组相比,Hcy组足细胞凋亡水平增加(P<0.05),而miR-30a表达水平降低(P<0.01);同时,miR-30a启动子区DNA甲基化程度升高(P<0.05);感染miR-30a mimic后,足细胞损伤明显减轻,凋亡形态的细胞数量减少,而感染miR-30a inhibitor后足细胞损伤程度加剧,凋亡形态的细胞数量增加。结论 miR-30a可抑制足细胞凋亡,其机制可能是miR-30a启动子区DNA低甲基化发挥重要作用。

Objective To investigate the effect of miR-30 a on homocysteine-induced podocytes apoptosis and the mechanism.Methods Glomerular podocytes were cultured in the absence(control group) or presence of 80 μmol·L^-1 homocysteine(Hcy) alone(Hcy group) for 48 h. The effects of Hcy on podocyte apoptosis were tested by using flow cytometry analysis and Annexin-V FITC dyeing. Validation of miR-30 a expression was performed by qRT-PCR, and nested methylation specific PCR(nMS-PCR) was used to assess the DNA methylation of miR-30 a promoter. Transmission electron microscopy was applied to observe the podocyte apoptosis after infected with miR-30 a mimic and miR-30 a inhibitor. Results Compared with the control group, Hcy significantly induced the apoptosis of podocytes(P<0.05), and the expression of miR-30 a was reduced(P<0.01) as the methylation of miR-30 a was increased(P<0.05). After transfection with miR-30 a mimic, podocyte injury was significantly reduced, and the apoptosis of podocytes was supressed. However, the podocyte injury and the number of apoptotic cells were increased by using miR-30 a inhibitor transfection. Conclusion Hypomethylation of miR-30 a may inhibit podocytes apoptosis.