MicroRNA-155在高糖诱导人血管内皮细胞中的表达及功能研究

The expression and function study of microRNA-155 in vascular endothelial cells apoptosis induced by high glucose

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 study the expression of microRNA-155（miR-155）in vascular endothelial cells treated with high glucose,and explore whether miR-155 participates in the process of cell apoptosis in vascular endothelial cells induced by high glucose through regulating the expression of PDCD4（Programmed cell death 4）.Method：HUVECs were treated with D-Glucose in 5.5mmol/L,11mmol/L,22mmol/L and 33mmol/L respectively for 48 hours.MiR-155,BCL-2associated X Protein（BAX）,B cell lymphoma/lewkmia（BCL-2）and Apoptosis-related cysteine peptidase（CASPASE-3）mRNA expressions were detected by Quantitative RT-PCR.The expressions of BAX,BCL-2 and CASPASE-3 were also determined by Western blot.The target gene of miR-155 was predicted by bioinformatics,and verified by dual luciferase reporter assay.After being transfected with the miR-155 mimics and negative control miRNA to HUVECs,the expression of target genes and the apoptosis rate of cells were assessed.At the same time,the apoptosis rate of cells was also observed by interfering the expression of target gene.Result：With the increasing of glucose concentrations,the levels of miR-155 and BCL-2mRNA expression in HUVECs cell were down-regulated in dose-dependent manner（P〈0.05）,the levels of BCL-2protein expression was also down regulated in dose-dependent manner（P〈0.05）;while the expression of BAX and CASPASE-3mRNA and protein in HUVECs cell were up-regulated in dose-dependent manner（P〈0.05）.PDCD4 was miR-155 targeted gene in HUVECs,which was testified by dual luciferase reporter assay.MiR-155 can down-regulated the expression of PDCD4 and decrease apoptosis rate of HUVECs cell,while PDCD4 can promote its apoptosis in vitro.Conclusion：High glucose can decrease miR-155 expression and increase the expression of apoptosis related genes.MiR-155 participates in the process of cell apoptosis in vascular endothelial cells induced by high glucose through regulating the expression of PDCD4.