miR-587调节TLR4表达对人主动脉内皮细胞生长的影响

miR-587 effect the growth of human aortic endothelial cells by regulating the expression of TLR4

目的探讨miR-587是如何通过调节TLR4的表达来影响人主动脉内皮细胞生长的。方法通过real-time PCR分析ox-LDL诱导/非诱导的人主动脉内皮细胞中miR-587和TLR4的表达。MTT实验检测miR-587对细胞增殖的影响。通过JC-1实验分析miR-587对细胞凋亡的影响。MIRDB预测miR-587与TLR4的结合位点后,利用荧光素酶报告基因实验检测miR-587对TLR4的靶向作用,并通过Western blot实验检测miR-587对TLR4和Caspase 8的调节作用。在过表达miR-587的细胞中转染TLR4,通过MTT、JC-1和Western blot检测细胞生长、凋亡和TLR4和Caspase 8的表达情况。结果 real-time PCR指出ox-LDL诱导能够下调miR-587的表达并且促进TLR4的表达。MTT实验表明,转染miR-587后细胞的增殖受到了显著的促进。JC-1实验发现miR-587可以抑制人主动脉内皮细胞的凋亡。随后MIRDB预测发现miR-587与TLR4的3′UTR区域具有结合位点,荧光素酶报告基因实验证明了miR-587可以直接作用于TLR4。Western blot实验证明miR-587能够抑制TLR4和Caspase 8蛋白的表达。转染TLR4可以减弱过表达miR-587对细胞增殖的促进、凋亡的抑制以及TLR4和Caspase 8表达的下调。结论 miR-587可以通过抑制TLR4进而促进人主动脉内皮细胞的生长。

Objective To investigate how miR-587 affected the growth of human aortic endothelial cells by regulating the expression of TLR4.Methods Real-time PCR was used to analyze the expression of miR-587 and TLR4 in ox-LDL-induced/non-induced human aortic endothelial cells.MTT assay was used to detect the effect of miR-587 on cell proliferation.JC-1 assay was used to analyze the effect of miR-587 on apoptosis.After MIRDB predicting the binding site of miR-587 to TLR 4,luciferase reporter gene assay was used to detect the targeting effect of miR-587 on TLR 4,and western blot was used to assess the regulatory effect of miR-587 on TLR4 and caspase8.TLR4 was transfected into the miR-587 over-expressed cells.Cell growth,cell apoptosis and expression of TLR4 and caspase 8 were assessed by MTT,JC-1 and Western blot.Results Real-time PCR indicated that ox-LDL induction down-regulated the expression of miR-587 and promoted the expression of TLR4.MTT assay showed that the proliferation of cells transfected with miR-587 was significantly promoted.JC-1 experiment showed that miR-587 inhibited the apoptosis of human aortic endothelial cells.Subsequently,MIRDB predicted that there was a binding site between the 3'UTR region of miR-587 and TLR4.Luciferase reporter gene experiments showed that miR-587 could directly act on TLR4.Western blot experiments showed that miR-587 inhibited the expression of TLR4 and caspase8.Transfection of TLR4 reduced the proliferation and apoptosis of the c ells,so was the down-regulation of TLR4 and caspase-8.Conclusion miR-587 can promote the growth of human aortic endothelial cells by inhibiting TLR4.