MicroRNA-21及其靶基因程序性细胞死亡因子4在动脉粥样硬化中的作用

Roles of microRNA-21 and its target gene programmed cell death 4 in atherosclerosis

目的探讨microRNA(miR)-21及其靶基因程序性细胞死亡因子4(PDCD4)在动脉粥样硬化(AS)发生发展中的作用。方法收集38例急性ST段抬高型心肌梗死(STEMI)患者及34例非冠心病患者的血浆,实时定量PCR检测miR-21的表达水平。在Lipo3 000的介导下,将miR-21类似物、抑制剂及沉默PDCD4(siP DCD4)分别转染鼠巨噬细胞系RAW 264.7细胞,并用氧化修饰低密度脂蛋白(ox-LDL)与细胞共同孵育,设立空白对照组(未转染且未加ox-LDL)及ox-LDL组(仅加oxLDL)。采用油红O染色检测细胞泡沫化程度,实时定量PCR及Western blot分别检测泡沫细胞中miR-21及PDCD4蛋白水平。此外,建立ApoE^(-/-)小鼠AS模型20只,按随机数字表法分为3组:单纯高脂喂养组6只、过表达miR-21组7只及阴性对照组(NC,7只),鼠尾静脉注射胆固醇包裹的miR-21激动剂(agomiR-21)及阴性对照agomiR-NC分别建立过表达miR-21组及NC组。选用正常饲料喂养的C57BL/6小鼠作为正常对照组。实时定量PCR检测miR-21在AS斑块及正常动脉组织中的表达;Western blot及免疫组化分别检测PDCD4蛋白水平。结果与相应的对照组比较,miR-21在STEMI患者血浆、泡沫细胞及单纯高脂喂养ApoE^(-/-)小鼠斑块组织中的表达水平均明显升高(P<0.001;P<0.01;P<0.01);泡沫细胞及单纯高脂喂养ApoE^(-/-)小鼠斑块内PDCD4蛋白水平明显增多;与ox-LDL组比较,ox-LDL+miR-21类似物组及ox-LDL+siP DCD4组巨噬细胞泡沫化程度明显下降,ox-LDL+miR-21抑制剂组细胞泡沫化程度明显增加。过表达miR-21组AS斑块内PDCD4蛋白水平较NC组明显减少,且斑块较NC组明显减小。结论 miR-21在急性STEMI患者血浆中、泡沫细胞及AS斑块内的水平明显升高。过表达miR-21及沉默PDCD4可抑制巨噬细胞向泡沫细胞的转化,这与动物组织实验结果一致,表明miR-21或可靶向调控PDCD4的表达,从而发挥抗AS的作用。

Objective To investigate the roles of microRNA（ miR）-21 and its target gene,programmed cell death 4（ PDCD4） in the development of atherosclerosis（ AS）. Methods The plasma of38 patients with acute ST elevation myocardial infarction（ STEMI） and 34 non-coronary heart disease patients were collected and used for detecting the levels of miR-21 by real-time quantitative PCR. Rat macrophage line RAW 264. 7 cells were transfected with the miR-21 analogue,inhibitor and silencing PDCD4（ siP DCD4） in mediation of Lipo3000,then co-incubated with ox-LDL and cultured for 48 h,blank control group（ untransfected with no ox-LDL） and ox-LDL group（ ox-LDL only） were also set up meanwhile. The effect of miR-21 and PDCD4 on foam RAW 264. 7 cells was analyzed by oil red staining.High fat feeding was given to 20 ApoE^（-/-）male mice（ 4-6 weeks） to establish AS animal models,and the mice were randomly divided into 3 groups： 6 in AS group,7 in miR-21 mimics treated group and 7 in NC group. Every 3 days,mice in the miR-21 mimics treated group and NC group received tail vein injection of cholesterol conjugated miR-21 agonist（ agomiR-21） and agomiR-NC,respectively. C57BL/6 mice fed with standard diet were arranged in the normal control group. qRT-PCR was performed to detect the expression levels of miR-21 in atherosclerotic plaque and normal artery. Immunohistochemistry and Western blot were used to measure the expression of PDCD4 protein. Results The expression levels of miR-21 in plasma of patients with STEMI,foam cells and plaque tissue from simple high fat fed ApoE^（-/-）mouse were significantly higher than those in the control group（ P 0. 001,P 0. 01,P 0. 01,respectively）. As compared with those in ox-LDL group,the bubbling levels in ox-LDL ＋ miR-21 mimics group and ox-LDL ＋ siP DCD4 group decreased significantly while those in ox-LDL ＋ miR-21 inhibitor group increased significantly. Compared with the C57BL/6 mice,the high fat diet-fed ApoE^（-/-）mice exhibited a greater accumulation of PDCD4 protein in the aorta. Nevertheless,tail vein injection of miR-21 mimics markedly inhibited the protein expression of PDCD4,and significantly decreased the sizes of AS areas. Conclusions Overexpression of miR-21 may inhibit the transformation of macrophages to foam cells and decrease AS area by regulating the expressions of PDCD4 thus plays a critical and protective role in anti-AS.