摘要
目的探讨miR-206在香烟烟雾提取物(CSE)诱导人肺微血管内皮细胞(HPMECs)凋亡中的分子作用机制。方法实时定量PCR检测相关基因的表达水平;Western blot检测相关蛋白的表达水平;流式细胞技术检测细胞凋亡;萤光素酶实验验证miR-206与下游基因的结合位点。结果不同浓度的CSE (0.5%～5.0%)处理能够促进HPMECs凋亡的同时增加miR-206的表达水平(P<0.05)。MiR-206过表达促进HPMECs的凋亡,同时提高cleaved caspase-3和caspase-9蛋白水平(P<0.05);而miR-206低表达可以抑制CSE引起的HPMECs凋亡以及凋亡相关蛋白(cleaved caspase-3和caspase-9)表达的上调(P<0.05)。生物信息学分析以及萤光素酶实验结果证实miR-206可以通过作用于notch3 3'非翻译区从而抑制notch3的表达。进一步的营救实验发现,notch3过表达可以拮抗miR-206引起的HPMEC凋亡。结论 MiR-206在CSE处理的HPMECs中表达上调,miR-206低表达对CSE引起的凋亡起到抑制作用,这种作用可能是通过靶向调控notch3来实现的。
Objective To explore the molecular mechanisms of miR-206 in the apoptosis of human pulmonary microvascular endothelial cell（HPMEC） induced by cigarette smoke extract（CSE）. Methods Relevant gene expression was determined by qRT-PCR; related protein levels were detected by western blot assay; cell apoptosis was measured by flow cytometry; the binding site of miR-206 and downstream genes was verified by luciferase reporter assay.Results Treatment with different concentrations of CSE（0.5%-5.0%） promoted the apoptosis of HPMECs and increase the expression level of miR-206（P〈0.05）. Overexpression of miR-206 promoted HPMECs apoptosis and increased the protein levels of cleaved caspase-3 and caspase-9（P〈0.05）. While down-expression of miR-206 inhibited CSE-induced HPMEC apoptosis and up-regulated expression of apoptosis-related proteins（cleaved caspase-3 and caspase-9）（P〈0.05）. Bioinformatics analysis and luciferase reporter assay results confirmed that miR-206 negatively regulated the expression of notch3 by targeting on 3＇untranslated region of notch3（P〈0.05）. Further rescue experiments showed that notch3 overexpression antagonized miR-206 induced HPMEC apoptosis（P〈0.05）. Conclusion MiR-206 is up-regulated expression in CSE-treated HMPEMCs, and down-regulation of miR-206 attenuates CSE-induced apoptosis, which may be achieved by targeted regulation of notch3.
作者
任佳荣
任登华
徐晶晶
黄永刚
REN Jia-rong;REN Deng-hua;XU Jing-jing;HUANG Yong-gang(Department of Respiratory Medicine,Wuxi Xishan People＇s Hospital,Zhongda Hospital Southeast University（Wuxi Branch）,Wuxi 214011,Jiangsu,CHINA)
出处
《海南医学》
CAS
2018年第19期2670-2674,共5页
Hainan Medical Journal
