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目的:研究microRNA-155(miR-155)高糖诱导人血管内皮细胞(HUVECs)中的表达,并探讨miR-155是否通过调节PDCD4(Programmed cell death 4)的表达参与了高糖诱导的血管内皮细胞凋亡的过程。方法:用不同浓度D-葡萄糖(5.5、11、22及33mmol/L)孵育HUVECs 48h,采用荧光定量PCR技术(Quantitative RTPCR)检测细胞中miR-155、BAX(BCL-2associated X Protein)、BCL-2(B cell lymphoma/lewkmia)及CASPASE-3(Apoptosis-related cysteine peptidase)的mRNA表达情况;采用Western blot方法检测BAX、BCL-2及CASPASE-3蛋白在细胞中的表达变化。通过数据库及生物信息学软件预测miR-155的靶基因,并利用双荧光素酶报告基因系统进行验证。进一步通过转染miR-155mimics和阴性对照miRNA至HUVECs后观察其对靶基因表达及细胞凋亡功能的影响;同时在HUVECs细胞中干扰靶基因的表达并观察其对细胞凋亡功能的影响。结果:随着葡萄糖浓度的增加,HUVECs中miR-155与BCL-2的mRNA表达水平呈浓度依赖性降低(P<0.05),BCL-2的蛋白表达水平也呈浓度依赖性降低(P<0.05);而BAX和CASPASE-3的mRNA及蛋白表达水平呈浓度依赖性升高(P<0.05)。生物信息学分析发现miR-155的靶基因为PDCD4,经体外双荧光素酶报告基因系统检测,证实PDCD4是miR-155的靶基因。体外细胞实验发现miR-155能降低靶基因PDCD4的mRNA与蛋白的表达水平,并抑制HUVECs细胞的凋亡;PDCD4具有促进HUVECs细胞凋亡的功能。结论:高浓度葡萄糖可降低血管内皮细胞中miR-155的表达水平,并增加凋亡相关基因的表达水平。PDCD4是血管内皮细胞中miR-155的重要靶基因,miR-155通过调节PDCD4表达参与了高糖诱导的血管内皮细胞的凋亡过程。
Objective: To investigate the expression of microRNA-155 (miR-155) in human vascular endothelial cells (HUVECs) induced by high glucose and to explore whether miR-155 participates in the hyperglycemia-induced vascular endothelial cell death by regulating the expression of PDCD4 The process of apoptosis. Methods: HUVECs were incubated with different concentrations of D-glucose (5.5, 11, 22 and 33 mmol / L) for 48 h. Quantitative RT-PCR was used to detect the expression of miR-155, BAX- 2 (B cell lymphoma / lewkmia) and CASPASE-3 (Apoptosis-related cysteine peptidase). Western blot was used to detect the expression of BAX, BCL-2 and CASPASE-3 in cells. The target genes of miR-155 were predicted by database and bioinformatics software and verified by dual luciferase reporter system. Furthermore, miR-155mimics and negative control miRNAs were transfected into HUVECs to observe their effect on target gene expression and apoptosis. Meanwhile, the expression of target genes was also interfered in HUVECs and their effects on apoptosis were observed. Results: With the increase of glucose concentration, the expression of miR-155 and BCL-2 in HUVECs was decreased in a concentration-dependent manner (P <0.05), and the protein expression of BCL-2 was also decreased in a concentration-dependent manner ), While BAX and CASPASE-3 mRNA and protein expression in a concentration-dependent manner (P <0.05). Bioinformatics analysis revealed that the target gene of miR-155 was PDCD4, which was detected by in vitro dual luciferase reporter gene system and confirmed that PDCD4 is the target gene of miR-155. In vitro experiments showed that miR-155 can reduce the expression of PDCD4 mRNA and protein and inhibit the apoptosis of HUVECs. PDCD4 can promote the apoptosis of HUVECs. Conclusion: High concentration glucose can decrease the expression of miR-155 in vascular endothelial cells and increase the expression of apoptosis-related genes. PDCD4 is an important target gene of miR-155 in vascular endothelial cells. MiR-155 is involved in the apoptosis of vascular endothelial cells induced by high glucose by regulating PDCD4 expression.