MEF 2A基因沉默对小鼠主动脉内皮细胞ICAM-1、VCAM-1表达的影响

Influence of MEF 2A gene silencing on expressions of ICAM-1 and VCAM-1 in mouse aortic endothelial cells

目的探讨肌细胞增强因子2A(MEF 2A)基因沉默对小鼠主动脉内皮细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)及纤溶酶原激活物抑制剂-1(PAI-1)表达的影响。方法建立MEF 2A基因沉默慢病毒转染体系,另外体外培养Apo E-/-小鼠主动脉内皮细胞。分为MACE细胞空白对照组、MACE细胞慢病毒阴性对照组、MACE细胞MEF 2A RNAi A、B、C、D组(选择沉默率最高的靶点),Apo E-/-小鼠主动脉内皮细胞组。采用RT-PCR、ELISA和Western blot方法检测各组细胞MEF 2A、ICAM-1、VCAM-1和PAI-1表达情况。结果 Apo E-/-小鼠主动脉内皮细胞MEF 2A m RNA表达水平明显高于MACE空白对照组细胞(P<0.05)。MEF 2A基因B位点沉默率最高,MEF 2A m RNA表达水平明显低于其他各组细胞,与MACE空白对照组相比,差异具有统计学意义(P<0.05)。经ELISA、RT-PCR和western blot检测结果显示,MEF 2A RNAi的MACE细胞组细胞和Apo E-/-小鼠主动脉内皮细胞中ICAM-1、VCAM-1、PAI-1蛋白和m RNA表达水平分别较空白对照组和阴性对照组明显上调(P<0.05)。MEF 2A表达量与ICAM-l、VCAM-1和PAI-1的表达量呈现负相关性(r=-0.985,-0.805,-0.959,P均<0.05)。结论 MEF 2A在动脉粥样硬化小鼠主动脉内皮细胞中表达降低,MEF 2A基因沉默可明显上调ICAM-1、VCAM-1、PAI-1的表达。

Objective To investigate the influence of myocyte enhancer factor 2A(MEF 2A)gene silencing in MACE cells by RNA interference(RNAi)on expressions of intercellular adhesion molecule-1(ICAM-1),vascular cell adhesion molecules-1(VCAM-1)and plasminogen activator inhibitor-1(PAI-1)in mouse aortic endothelial cells(AEC).Methods A lentiviral transfection system of MEF 2A gene silencing was established in MACE cells,and mouse AEC were in vitro cultured in ApoE-/-mice.The MACE cells were divided into blank control group,negative control group,MEF 2A RNAi groups A,B,C and D(selected the target with the highest silencing rate),and mouse AEC were included into mouse AEC group.The expressions of MEF 2A,ICAM-1,VCAM-1 and PAI-1 were detected by using RT-PCR,ELISA and Western blotting assay.Results The expression of MEF 2A mRNA was significantly higher in mouse AEC group than that in blank control group(P<0.05).The silencing rate was the highest and expression of MEF 2A mRNA was significantly lower than that in other groups at gene site B of MEF 2A,and difference had statistical significance compared with blank control group(P<0.05).The results of ELISA,RT-PCR and Western blotting assay showed that protein and mRNA expressions of ICAM-1,VCAM-1 and PAI-1 were significantly up-regulated in MEF 2A RNAi groups A,B,C and D and mouse AEC group compared with blank control group and negative control group(P<0.05).The expression of MEF 2A was negatively correlated to expressions of ICAM-1,VCAM-1 and PAI-1(r=-0.985,-0.805,-0.959,all P<0.05).Conclusion The expression of MEF 2A decreases in mouse AEC in mice with atherosclerosis.MEF 2A gene silencing can significantly upregulated the expressions of ICAM-1,VCAM-1 and PAI-1.