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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(30mmol/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。
AIM: To investigate the effect of miR-217 on endothelial cell apoptosis induced by high glucose in endothelial cells. Methods: Human coronary artery endothelial cells were cultured and stimulated with culture medium containing D-glucose (30 mmol / L): (1) Real-time quantitative PCR was used to detect the expression of miR-217, miR- miR-218, miR-451, miR-328, miR-517c and miR-216a, etc.); (2) Using lentivirus infection to intervene the level of miR-217 in endothelial cells and detect apoptosis by flow cytometry (3) bioinformatics prediction, dual luciferase reporter gene validation and western blot to determine the target gene of miR-217. Results: (1) The expression of miR-217, miR-137, miR-29c and miR-218 were up-regulated after high glucose stimulation in real- The expression of miR-517c and miR-216a were down-regulated (P <0.01), and miR-217 was 5.67-fold more than that of control cells. (2) After lentivirus infection of endothelial cells and then stimulated by high glucose, the results of flow cytometry (3) Bioinformatics analysis showed that there was a miR-217 binding site on the 3 ’untranslated region of SIRT1 gene, the dual luciferase reporter gene and the western The result of blot showed that SIRT1 is the target gene of miR-217. Conclusion: miR-217 may be involved in the regulation of apoptosis induced by high glucose by inhibiting the expression of SIRT1.