抑制长链非编码RNA MALAT1对糖脂毒性诱导的内皮细胞功能障碍的影响及可能机制  

Effect and Potential Mechanism of Inhibition of Long Non-coding RNA MALAT1 on Glycolipipotoxicityinduced Endothelial Cell Dysfunction

作　　者：张志扬 刘芬[2] 张雪鹤 房彬彬[2] 张冀鑫 谢骞 杨毅宁 李晓梅[1] ZHANG Zhiyang;LIU Fen;ZHANG Xuehe;FANG Binbin;ZHANG Jixin;XIE Qian;YANG Yining;LI Xiaomei(Heart Center,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China;Key Laboratory of Pathogenesis,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China;Department of Cardiology,People's Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830001,China)

机构地区：[1]新疆医科大学第一附属医院心脏中心,乌鲁木齐830054 [2]新疆医科大学第一附属医院心血管重点实验室,乌鲁木齐830054 [3]新疆维吾尔自治区人民医院心内科,乌鲁木齐830001

出　　处：《中国循环杂志》2024年第2期185-193,共9页Chinese Circulation Journal

基　　金：国家自然科学基金项目(8216020109);中央引导地方计划项目(ZYYD2022C21);新疆维吾尔自治区重点研发项目(2020B03002-1);新疆维吾尔自治区重点研发项目(2020B03002-3)。

摘　　要：目的:探讨糖脂毒性环境中抑制长链非编码RNA(lnc RNA)人类肺腺癌转移相关转录本1(MALAT1)的表达水平对人脐静脉内皮细胞功能影响的分子机制。方法:用葡萄糖和棕榈酸处理人脐静脉内皮细胞,建立体外糖脂毒性内皮细胞模型,并分为对照组、高糖高脂组、高糖高脂对照组以及小干扰RNA(si-RNA)干预的高糖高脂+si-MALAT1组、高糖高脂+si-丝裂原活化蛋白激酶1(si-MAPK1)组。应用实时荧光定量PCR检测MALAT1、MAPK1的mRNA表达水平;蛋白免疫印迹法检测自噬、线粒体融合分裂、凋亡、通路相关蛋白的表达水平;免疫荧光共聚焦定位检测自噬及溶酶体相关蛋白的荧光共定位情况;透射电镜观察内皮细胞中自噬溶酶体的数目;线粒体探针染色检测线粒体形态;免疫荧光检测细胞内活性氧(ROS)的产生;流式细胞仪检测各组细胞的凋亡;细胞增殖及划痕实验检测不同时间点各组细胞的增殖及迁移能力;血管形成试验检测各组细胞中新生血管数量。结果:与对照组相比,高糖高脂组、高糖高脂对照组的MALAT1 mRNA、磷酸化的丝裂原活化蛋白激酶1(p-MAPK1)的表达水平增加,磷酸化的哺乳动物雷帕霉素靶蛋白(p-mTOR)的表达水平下降,P均<0.05。与高糖高脂对照组相比,高糖高脂+si-MALAT1组微管相关蛋白1A/1B-轻链3(LC3)、螯合体1(p62)、ROS、剪切后活化的半胱氨酸天冬氨酸蛋白水解酶-3(cleaved caspase-3)、BCL-2相关X蛋白(BAX)、p-MAPK1的表达水平均下降,线粒体融合蛋白视神经萎缩蛋白1(OPA1)、BCL-2、p-mTOR的表达水平均增加,P均<0.05;LC3和溶酶体相关膜蛋白2(LAMP2)蛋白的荧光共定位阳性颗粒增加(P均<0.01),溶酶体数目减少;细胞增殖、迁移、成管能力均增加(P均<0.01)。与高糖高脂对照组相比,高糖高脂+si-MAPK1组内皮细胞中p-MAPK1的表达下降,p-mTOR的表达上升(P均<0.01)。结论:抑制MALAT1表达,可降低糖脂毒性环境中线粒体的自噬水�Objectives:To investigate the effect of inhibition of long non-coding RNA(lnc RNA)in human metastasis associated lung adenocarcinoma transcript 1(MALAT1)on glycolipitoxicity-induced human umbilical vein endothelial cell dysfunction.Methods:Human umbilical vein endothelial cells were treated with glucose and palmitic acid in vitro to establish the glycolipitoxic endothelial cell models.Following groups were examined:control group,high-glucose and high-fat group,high-glucose and high-fat+non-targeting RAN control group,high-glucose and high-lipid+MALAT1 siRNA group,and highglucose and high-lipid+MAPK1 siRNA group.RT-qPCR was used to detect the mRNA expression of MALAT1 and MAPK1.Western blot was used to detect the expression levels of autophagy,mitochondrial fusion division,apoptosis,and pathwayrelated proteins.Immunofluorescence confocal localization was used to detect the fluorescence colocalization of autophagy and lysosome-related proteins.The number of autophagolysosomes in endothelial cells was observed by transmission electron microscopy.Mitochondrial probe staining was used to detect mitochondrial morphology,immunofluorescence was used to detect intracellular reactive oxygen species(ROS)production,flow cytometry was used to detect the apoptosis of cells in each group,cell proliferation and scratch assays were used to detect the proliferation and migration ability of cells in different groups at different time points.The angiogenesis was quantified by counting the number of new blood vessels in each group.Results:Compared with the control group,the expression of lncRNA MALAT1 mRNA and the expression of phosphorylated mito-activated protein kinase 1(p-MAPK1)were upregulated(both P<0.05)and the expression of phosphorylated mammalian target protein(p-mTOR)was downregulated in the high-glucose and high-fat group and the highsugar and high-fat control group(all P<0.01).Compared with the high-glucose and high-fat non-targeting RNA control group,the expressions of microtubule-associated protein 1A/1B-light chain 3(

关 键 词：长链非编码RNA人类肺腺癌转移相关转录本1 丝裂原活化蛋白激酶1 哺乳动物雷帕霉素靶蛋白 糖脂毒性 内皮细胞功能 线粒体自噬 凋亡 

分 类 号：R54[医药卫生—心血管疾病]