N6-甲基腺苷甲基化转移酶3对动脉粥样硬化的影响及其作用机制  

The effect of methyltransferase like 3 on atherosclerosis and its mechanism of action

作　　者：种泓昭 张昊 朱坤鹏 张大伟[2] Zhong Hongzhao;Zhang Hao;Zhu Kunpeng;Zhang Dawei(The First Clinical Medical College of Shandong University of Traditional Chinese Medicine,Jinan 250000,China;Department of Vascular Surgery,Affiliated Hospital of Shandong University of Traditional Chinese MMeodicine,Jinann 225500000000,China)

机构地区：[1]山东中医药大学第一临床医学院,济南250000 [2]山东中医药大学附属医院血管外科,济南250000

出　　处：《中华实验外科杂志》2024年第10期2226-2229,共4页Chinese Journal of Experimental Surgery

基　　金：山东省中医药科技发展计划(2019-0203)。

摘　　要：目的:探讨N6-甲基腺苷甲基化转移酶3(METTL3)调节动脉粥样硬化的作用及其分子机制。方法:人THP-1单核细胞采用100 ng/ml PMA和75μg/ml Ac-LDL诱导贴壁,采用荧光定量聚合酶链反应(PCR)和蛋白质免疫印迹分析METTL3和肝癌衍生生长因子(HDGF)mRNA和蛋白质表达水平。采用短发卡RNA(shRNA)敲低和过表达METTL3,分析肝癌衍生生长因子(HDGF)mRNA和蛋白质表达水平;生物信息学分析mRNA上m6A修饰情况;Dil-Ac-LDL荧光探针分析巨噬细胞脂滴摄取能力。高脂喂养的ApoE-/-小鼠分为METTL3/对照组和METTL3/HDGF敲低组,采用油红O染色分析主动脉脂质堆积情况,采用苏木精-伊红(HE)染色分析动脉粥样硬化斑块面积。组间计量数据比较采用独立样本t检验。结果:THP-1巨噬细胞经75μg/ml Ac-LDL处理后,对照组细胞METTL3和HDGF mRNA和蛋白质(1.03±0.09、0.60±0.09;1.00±0.08、0.76±0.13)明显低于泡沫化组(1.68±0.11、1.01±0.11;1.58±0.11、1.19±0.08),差异有统计学意义(t=12.920、8.263、12.470、7.746,P<0.05)。对照组巨噬细胞HDGF mRNA和蛋白质(1.72±0.14、1.18±0.08)明显高于METTL3 KD组(1.01±0.10、0.72±0.15),差异有统计学意义(t=11.850、7.875,P<0.05)。对照组巨噬细胞胆固醇提呈率(0.48±0.06)明显高于METTL3 KD组(0.24±0.04),差异有统计学意义(t=9.155,P<0.05)。对照组巨噬细胞胆固醇提呈率[(329.08±23.15)mg/ml]明显高于METTL3 KD组[(236.08±21.53)mg/ml],差异有统计学意义(t=8.319,P<0.05)。对照组巨噬细胞HDGF mRNA m6A水平(1.00±0.09)明显高于METTL3 KD组(0.53±0.12),差异有统计学意义(t=8.799,P<0.05)。METTL3/对照组小鼠总动脂质沉积比和主动脉斑块面积比[(35.52±5.87)%、0.65±0.06]明显高于METTL3/HDGF敲低组[(22.96±3.43)%、0.29±0.05],差异有统计学意义(t=5.227、12.090,P<0.05)。结论:m6A甲基化酶METTL3通过RNA甲基化上调HDGF表达,促进巨噬细胞脂质大量堆积,加剧动脉粥样硬化的进展。Objective:To investigate the role and molecular mechanism of methyltransferase like 3(METTL3),an N6-methyladenosine(m6A)methyltransferase,in regulating atherosclerosis.Methods:Human THP-1 monocytes were induced to adhere by incubation with 100 ng/ml PMA and 75μg/ml Ac-LDL.The expression levels of METTL3 and hepatoma-derived growth,factor(HDGF)mRNA and protein were analyzed by quantitative polymerase chain reaction(PCR)and protein immunoblotting.METTL3 was knocked down or overexpressed,and the expression levels of HDGF mRNA and protein were analyzed.mRNA m6A modification was analyzed by m6A methylation immunoprecipitation sequencing.The ability of macrophages to take up lipid droplets was analyzed by Dil-Ac-LDL fluorescent probe.ApoE-/-mice were fed a high-fat diet and divided into METTL3/control and METTL3/HDGF-KD groups.The degree of atherosclerotic lipid accumulation in the aorta was analyzed by oil red O staining.The area of atherosclerotic plaques was analyzed by hematoxylin and eosin(HE)staining and Masson staining.Intergroup comparisons of quantitative data were made using independent sample t-tests.Results:After Ac-LDL treatment of 75μg/ml for THP-1 macrophages,the expression levels of METTL3 and HDGF mRNA and protein in the control group were significantly lower than those in the foam cell group(1.03±0.09,0.60±0.09;1.00±0.08,0.76±0.13,t=12.920,8.263,12.470,7.746,P<0.05),while the expression levels of HDGF mRNA and protein in the METTL3 KD group were significantly higher than those in the control group(1.72±0.14,1.18±0.08,t=11.850,7.875,P<0.05).The cholesterol uptake rate of macrophages in the control group(0.48±0.06)was significantly higher than that of the METTL3 KD group macrophages(0.24±0.04)(t=9.155,P<0.05).The cholesterol uptake rate of macrophages in the control group[(329.08±23.15)mg/ml]was significantly higher than that of the METTL3 KD group macrophages[(236.08±21.53)mg/ml,t=8.319,P<0.05].The m6A level of HDGF mRNA in the control group macrophages(1.00±0.09)was significantly higher t

关 键 词：N6-甲基腺苷甲基化酶3 动脉粥样硬化 甲基化 

分 类 号：R54[医药卫生—心血管疾病]