标题：MiR-206通过靶向调控notch3参与香烟烟雾诱导的人肺微血管内皮细胞凋亡

      作者：任佳荣，任登华，徐晶晶，黄永刚    (东南大学附属中大医院无锡分院无锡锡山人民医院呼吸内科，江苏 无锡 214011)

      卷次： 2018年29卷19期

      【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。
      【关键词】 miR-206；香烟烟雾提取物；人肺微血管内皮细胞；凋亡；notch3
      【中图分类号】 R329.2+8 【文献标识码】 A 【文章编号】 1003—6350（2018）19—2670—05

MiR-206 participates in cigarette smoke extract-induced apoptosis of human pulmonary microvascularendothelial cells via targeting regulation of notch3.
REN Jia-rong, REN Deng-hua, XU Jing-jing, HUANGYong-gang. Department of Respiratory Medicine, Wuxi Xishan People's Hospital, Zhongda Hospital Southeast University(Wuxi Branch), Wuxi 214011, Jiangsu, CHINA
【Abstract】 Objective To explore the molecular mechanisms of miR-206 in the apoptosis of human pulmonarymicrovascular endothelial cell (HPMEC) induced by cigarette smoke extract (CSE). Methods Relevant gene expres-sion was determined by qRT-PCR; related protein levels were detected by western blot assay; cell apoptosis was mea-sured 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 in-crease the expression level of miR-206 (P<0.05). Overexpression of miR-206 promoted HPMECs apoptosis and in-creased the protein levels of cleaved caspase-3 and caspase-9 (P<0.05). While down-expression of miR-206 inhibitedCSE-induced HPMEC apoptosis and up-regulated expression of apoptosis-related proteins (cleaved caspase-3 and cas-pase-9) (P<0.05). Bioinformatics analysis and luciferase reporter assay results confirmed that miR-206 negatively regu-lated the expression of notch3 by targeting on 3'untranslated region of notch3 (P<0.05). Further rescue experimentsshowed that notch3 overexpression antagonized miR-206 induced HPMEC apoptosis (P<0.05). Conclusion MiR-206is up-regulated expression in CSE-treated HMPEMCs, and down-regulation of miR-206 attenuates CSE-induced apopto-sis, which may be achieved by targeted regulation of notch3.
      【Key words】 miR-206; Cigarette smoke extract; Human pulmonary microvascular endothelial cell; Apoptosis;Notch3doi:10.3969/j.issn.1003-6350.2018.19.002