miR-218在高浓度葡萄糖诱导大鼠视网膜微血管内皮细胞凋亡中的作用

目的　探讨miR-218在高浓度葡萄糖诱导大鼠视网膜微血管内皮细胞（rat retinal microvascular endothelial cells，rRMECs）凋亡中的作用及其相关机制。方法　将rRMECs分为对照组、高糖组、miR-218阴性抑制组和miR-218抑制组。对照组细胞用DMEM低糖培养基培养；高糖组在培养基中添加D-葡萄糖，终浓度为25 mmol·L^-1；miR-218抑制组及miR-218阴性抑制组是在高糖组培养基的基础上，按照说明书操作，利用Lipo2000将miR-218抑制剂和阴性抑制剂转染rRMECs，共同孵育24 h。提取各组rRMECs总RNA并分析miR-218含量，检测细胞活力，计算细胞凋亡率以及采用Western blot检测各组rRMECs Caspase-3蛋白表达，利用SPSS 19.0统计学软件对实验数据进行单因素方差分析并进行两两比较。结果　对照组、高糖组、miR-218阴性抑制组、miR-218抑制组miR-218相对表达量分别为1.00±0.08、1.48±0.12、1.46±0.14、1.03±0.08；细胞活力分别为100.0%、（72.3±7.5）%、（70.7±9.3）%及（98.4±9.4）%；与对照组相比，高糖组和miR-218阴性抑制组的miR-218相对表达量明显升高（均为P<0.05），miR-218抑制组的miR-218相对表达量较对照组变化不显著（P>0.05）；高糖组和miR-218阴性抑制组rRMECs细胞活力与对照组相比均明显降低（均为P<0.05），miR-218抑制组与对照组相比细胞活力未见明显变化（P>0.05）。对照组、高糖组、miR-218阴性抑制组、miR-218抑制组rRMECs细胞凋亡率分别为（2.3±0.0）%、（20.3±3.1）%、（23.1±3.2）%及（2.9±0.1）%；高糖组和miR-218阴性抑制组的细胞凋亡率均显著高于对照组（均为P<0.05）；而miR-218抑制组与对照组相比，细胞凋亡率变化不明显，差异无统计学意义（P>0.05）。对照组rRMECs Caspase-3蛋白相对表达量为（18.6±2.3）%、高糖组为（43.3±4.5）%、miR-218阴性抑制组为（41.6±3.9）%、miR-218抑制组为（20.0±2.8）%。与对照组相比，高糖组和miR-218阴性抑制组的Caspase-3蛋白相对表达量明显增加（均为P<0.05），miR-218抑制组Caspase-3蛋白相对表达量变化不明显（P>0.05）。结论　高浓度葡萄糖能促使rRMECs miR-218相对表达量增高；当miR-218相对表达量增高时，细胞凋亡明显，细胞活力显著下降，细胞内Caspase-3蛋白的相对表达量明显上调。

Role of miR-218 in apoptosis of rat retinal microvascular endothelial cells induced by high concentration glucose

Objective　To explore the role of miR-218 in the apoptosis of rat retinal microvascular endothelial cells (rRMECs) induced by high concentration of glucose and its related mechanisms.Methods　The rRMECs were divided into control group, high glucose group, miR-218 negative inhibition group and miR-218 inhibition group. Cells in the control group were cultured with DMEM low-sugar medium; the high glucose group added D-glucose to the medium, and the final concentration was 25 mmol·L^-1; based on the medium of the high glucose group, Lipo2000 was used to transfect the miR-218 inhibitor and negative control inhibitor into rRMECs following the instructions in the miR-218 inhibition group and miR-218 negative inhibition group for 24 hours. We extracted the total RNA of rRMECs in each group for analyzing the content of miR-218, checking cell viability, and calculating the cell apoptosis rate. Western blot was applied to detect the expression of rRMECs Caspase-3 protein in each group, and SPSS 19.0 statistical software was used to perform one-way analysis of variance on the experimental data for making pairwise comparisons.Results　The relative expression levels of miR-218 in the control group, high glucose group, miR-218 negative inhibition group, and miR-218 inhibition group were 1.00±0.08, 1.48±0.12, 1.46±0.14, 1.03±0.08, respectively, and the cell viability was 100.0%, (72.3±7.5)%, (70.7±9.3)% and (98.4±9.4)%,respectively. Compared with the control group, the content of miR-218 in the high glucose group and the miR-218 negative inhibition group increased significantly (both P<0.05). Compared with the control group, the miR-218 content in the miR-218 inhibition group did not change significantly (P>0.05); compared with the control group, the cell viability of rRMECs in the high glucose group and miR-218 negative inhibition group was significantly lower (both P<0.05), compared with the control group, the cell viability of the miR-218 inhibition group did not change significantly (P>0.05). The apoptosis rate of rRMECs in the control group, high glucose group, miR-218 negative inhibition group, and miR-218 inhibition group were (2.3±0.0)%, (20.3±3.1)%, (23.1±3.2)% and (2.9±0.1)%, respectively. The apoptosis rate of high glucose group and miR-218 negative inhibition group was significantly higher than that of control group (both P<0.05); compared with the control group, the rate of cell apoptosis does not change significantly in the miR-218 inhibition group, and the difference was not statistically significant (P>0.05). The relative expression of rRMECs Caspase-3 was (18.6±2.3)%, (43.3±4.5)%, (41.6±3.9)%, and (20.0±2.8)% in the control group, high glucose group, miR-218 negative inhibition group, and miR-218 inhibition group, respectively. Compared with the control group, the expression of Caspase-3 in the high glucose group and the miR-218 negative inhibition group increased significantly (both P<0.05), the expression of Caspase-3 in the miR-218 inhibition group did not change significantly (P>0.05).Conclusion　High concentration of glucose can increase the content of rRMECs miR-218; when the content of miR-218 increases, apoptosis is obvious, cell viability decreased significantly, and the expression of Caspase-3 in cells was significantly up-regulated.