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目的：研究miR-217 对高糖诱导的内皮刺激内皮细胞凋亡的作用。方法：培养人冠状动脉内皮细胞，用含D- 葡萄糖（30 mmol/L）的培养液刺激：(1) 利用实时定量PCR 检测内皮细胞相关微小RNA（miR-217、miR-137、miR-29c、miR-218、miR-451、 miR-328、miR-517c 和miR-216a等）的表达变化；(2)利用慢病毒感染技术干预内皮细胞miR-217 水平，利用流式细胞术检测细胞凋亡水平；(3)生物信息学预测、双荧光素酶报告基因验证以及蛋白免疫印迹法（western blot）确定miR-217 的靶基因。结果：(1)实时定量PCR 检测发现高糖刺激内皮细胞后，miR-217、miR-137、miR-29c、miR-218 等的表达上调（P<0.01），miR-451、miR-328、 miR-517c 和miR-216a的表达下调（P<0.01），其中miR-217 比对照细胞升高了5.67 倍；(2) 慢病毒感染内皮细胞后再经高糖刺激，流式细胞术检测发现过表达miR-217 的内皮细胞凋亡水平有显著提高；(3) 生物信息学分析发现SIRT1 基因的3' 非翻译区上存在一个miR-217 的结合位点，双荧光素酶报告基因和western blot 结果均证明SIRT1 是miR-217的靶基因。结论：miR-217 可能通过抑制SIRT1的表达参与高糖诱导的内皮细胞凋亡的调控。

英文摘要:

Objective:To study the effect of miR-217 on apoptosis of endothelial cell induced by high-glucose.Methods:Human coronary artery endothelial cells were culture by medium with 30 mmol/L D-glucose, and related microRNAs (miR-217, miR-137, miR-29c, miR-218, miR-451, miR-328, miR-517c and miR-216a) were detected by real-time PCR. The cell apoptosis level of endothelial cells overexpressed miR-217 by using lentivirus infection technology was investigated by flow cytometry. Then, the targeted gene of miR-217 was identified by using bioinformatics, dual-luciferase assay and western blot.Results:Real-time PCR assay showed that miR-217, miR-137, miR-29c and miR-218 were significantly increased (P<0.01) after high-glucose stimulating, while miR-451, miR-328, miR-517c and miR-216a were significantly decreased (P<0.01). Especially, the miR-217 level was increased 5.67-fold than that of control cell. After overexpressing miR-217 by using lentivirus infection, these cells apoptosis level was obviously increased. Bioinformatics prediction found a binding-site in 3' untranslated region of SIRT1, this site was a targeted bind site of miR-217 confirmed by dualluciferase assay and western blot.Conclusion:MiR-217 may involve in apoptosis regulation of high-glucose induced endothelial cell injury by inhibiting expression of SIRT1.

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