miR-29c通过调控SIRT1表达介导动脉粥样硬化氧化应激与炎症反应

miR-29c influence oxidative stress and inflammation in atherosclerosis by regulating the expression of SIRT1

目的研究miR-29c能否通过调控沉默信息调节因子2相关酶类1(SIRT1)的表达影响动脉粥样硬化(AS)进程中氧化应激及炎症反应。方法采用ApoE-/-基因敲除小鼠(ApoE-/-),经适应性饲养,于6周龄时以高脂饲料喂养,构建AS模型(AS组);以C57BL/6J小鼠为正常对照组,给予普通饲料喂养。12周后取主动脉,检测miR-29c表达以及肿瘤坏死因子-α(TNF-α)mRNA水平、活性氧(ROS)、烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶水平。培养人脐静脉内皮细胞(HUVEC),分别转染miR-29c mimic和antagomir,以氧化型低密度脂蛋白(oxLDL)(50μg/ml)进行刺激,检测各组TNF-α表达、ROS、NADPH氧化酶水平。此外,实时PCR和Western blot检测oxLDL诱导的内皮细胞损伤模型中SIRT1 mRNA及蛋白表达水平变化,并进一步检测正常细胞转染miR-29c mimic与antagomir后SIRT1 mRNA及蛋白表达水平。结果与正常组比较,AS组miR-29c表达及TNF-αmRNA、ROS、NADPH氧化酶水平均明显升高(P<0.05)。oxLDL刺激后,与对照组相比,转染miR-29c antagomir细胞中TNF-α、ROS、NADPH氧化酶水平均明显降低(P<0.05);与之相反,转染miR-29c mimic细胞中TNF-α、ROS、NADPH氧化酶水平明显升高(P<0.05)。此外,oxLDL刺激HUVEC后,细胞的SIRT1 mRNA与蛋白表达水平均有所降低(P<0.05)。然而,正常细胞转染miR-29c mimic和antagomir后结果显示在SIRT1 mRNA水平上差异无统计学意义(P>0.05),而SIRT1蛋白表达水平有差异,且差异有统计学意义(P<0.05),提示miR-29c对SIRT1的调控可能处于翻译后水平。结论miR-29c可下调SIRT1表达进而增强AS进程中氧化应激及炎症反应。

Objective To investigate the roles of miR-29c in regulating the expression of silent mating type information regulation 2 homolog-1(SIRT1)involved in atherosclerosis(AS)oxidative stress and inflammatory processes.Methods After adaptive feeding,ApoE-/-knockout mice(ApoE-/-)were fed with a high-fat diet to build the atherosclerosis model at 6 weeks of age.In addition,C57BL/6J mice was used as the normal control group and fed with normal feed.After 12 weeks,the expression of miR-29c,tumor necrosis factor-α(TNF-α)mRNA,reactive oxygen species(ROS)production and nicotinamide adenine dinucleotide phosphate(NADPH)oxidase activity were detected in mouse aorta.Meanwhile,human umbilical vein endothelial cells(HUVEC)were cultured in vitro,transfected with miR-29c mimic and antagomir respectively and stimulated with oxidized low density lipoprotein(oxLDL)(50μg/ml).The levels of TNF-α,ROS and NADPH oxidase activity were tested in each group.Besides,the expression of SIRT1 mRNA and protein were detected in HUVEC after the stimulation of oxLDL by real time PCR and Western blot,which were also determined in miR-29c mimic and antagomir group.Results The results showed that the expression of miR-29c,TNF-αmRNA,ROS and NADPH oxidase activity significantly increased in AS group compared with the normal group.Under the stimulation of oxLDL,compared with the control group,the levels of TNF-α,ROS and NADPH oxidase activity in the cells decreased significantly when treated with miR-29c antagomir.On the contrary,the levels of TNF-α,ROS and NADPH oxidase activity were significantly increased after miR-29c mimic treatment.In addition,the expression of SIRT1 mRNA and protein were reduced in HUVEC after the stimulation of oxLDL.However,compared with the control group,there was no statistical difference in SIRT1 mRNA level in the antagomir and mimic groups,but the expression of SIRT1 protein was significant difference in the antagomir and mimic groups.The above indicated that the modulation of SIRT1 induced by miR-29c was at the post-translational level.Conclusion miR-29c can down-regulate the expression of SIRT1 and enhance oxidative stress and inflammatory response in the process of AS.