基于PI3K/Akt/mTOR信号通路的miR-24调控ox-LDL诱导的HUVECs自噬机制研究  

Study on the regulation of ox-LDL induced autophagy mechanism in HUVECs by miR-24 based on PI3K/Akt/mTOR signaling pathway

作　　者：杨鹏 杨增艳 翟阳 周炜潜 罗雪兰 欧和生 YANG Peng;YANG Zengyan;ZHAI Yang;ZHOU Weiqian;LUO Xuelan;OU Hesheng(Department of Science and Technology,Guangxi International Zhuang Medical Hospital,Nanning 530201,Guangxi,China)

机构地区：[1]广西国际壮医医院科技部,广西南宁530201

出　　处：《右江医学》2024年第9期775-783,共9页Chinese Youjiang Medical Journal

基　　金：国家自然科学基金(82060655);广西国际壮医医院“青苗工程”人才培育项目(2022001);国家中医药管理局高水平中医药重点学科建设项目-少数民族药学(壮药学)(zyyzdxk-2023165);广西国际壮医医院2023年高层次人才队伍建设三年行动计划项目(GZCX20231203)。

摘　　要：目的探讨miR-24对氧化低密度脂蛋白(ox-LDL)诱导下的人脐静脉内皮细胞(HUVECs)自噬的影响及其相关机制,为进一步阐明miR-24在动脉粥样硬化(AS)中的作用提供理论依据。方法采用实时荧光定量PCR(qRT-PCR)检测miR-24的表达;应用蛋白免疫印迹法(western blot)和qRT-PCR法检测Beclin-1、LC3Ⅰ/LC3Ⅱ、p-mTOR、p-PI3K、p-Akt的蛋白和mRNA表达水平;应用透射电子显微镜技术检测细胞的自噬小体生成情况;应用四甲基偶氮唑盐(MTT)法、细胞划痕法、Caspase-3比色法和Hoechst 33258染色法分别检测细胞活性、迁移和凋亡情况。结果应用ox-LDL诱导HUVECs后,发现HUVECs中miR-24的表达显著降低(P<0.05)。miR-24过表达可明显抑制ox-LDL诱导的HUVECs自噬(P<0.05),而miR-24低表达则会增加ox-LDL诱导的HUVECs自噬(P<0.05)。miR-24过表达可显著降低Beclin-1的表达水平,上调LC3Ⅰ/LC3Ⅱ的水平(P<0.05),同时,miR-24过表达可显著促进p-PI3K、p-Akt和p-mTOR的表达(P<0.05)。此外,miR-24过表达显著抑制HUVECs的活力和迁移,增加Caspase-3活性并促进其凋亡(P<0.05)。结论miR-24的过表达可激活PI3K/Akt/mTOR信号通路而降低ox-LDL诱导的HUVECs的自噬水平并促进其凋亡,miR-24可能成为AS的潜在治疗新靶点。Objective To investigate the effect of miR-24 on autophagy in human umbilical vein endothelial cells(HUVECs)induced by oxidized low-density lipoprotein(ox-LDL)and its related mechanism,so as to provide a theoretical basis for further elucidating the role of miR-24 in atherosclerosis(AS).Methods Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression of miR-24;western blot and qRT-PCR were applied to detect the protein and mRNA expression levels of Beclin-1,LC3Ⅰ/LC3Ⅱ,p-mTOR,p-PI3K,and p-Akt;transmission electron microscopy was used to measure the formation of autophagosomes in cells;MTT assay,cell scratch assay,Caspase-3 colorimetric assay,and Hoechst 33258 staining were used to assess cell viability,migration,and apoptosis,respectively.Results After inducing HUVECs with ox-LDL,it was found that the expression of miR-24 in HUVECs was significantly reduced(P<0.05).Overexpression of miR-24 significantly inhibited autophagy induced by ox-LDL in HUVECs(P<0.05),while downregulation of miR-24 increased autophagy induced by ox-LDL in it(P<0.05).The overexpression of miR-24 could significantly reduce the expression levels of Beclin-1 and upregulate the levels of LC3Ⅰ/LC3Ⅱ(P<0.05).At the same time,the overexpression of miR-24 could significantly promote the expressions of p-PI3K,p-Akt,and p-mTOR(P<0.05).Furthermore,overexpression of miR-24 significantly inhibited the viability and migration of HUVECs,increased the activity of Caspase-3,and promoted apoptosis(P<0.05).Conclusion Overexpression of miR-24 can activate PI3K/Akt/mTOR signaling pathway,reduce the level of autophagy induced by ox-LDL in HUVECs,and promote apoptosis,which miR-24 may become a potential new therapeutic target for AS.

关 键 词：氧化低密度脂蛋白 人脐静脉内皮细胞 PI3K/Akt/mTOR信号通路 miR-24 自噬 凋亡 

分 类 号：R36.12[医药卫生—病理学] R544[医药卫生—基础医学]