摘要
目的探讨MicroRNA-124-3p(miR-124-3p)在肺炎链球菌(SP)感染诱导的肺泡上皮细胞(AEC)凋亡中的分子机制。方法体外培养A549细胞,使用Lipofectamine®2000将miR-124-3p模拟物(miR-124-3p mimic)、pcDNA3.1-肿瘤坏死因子受体相关因子6(TRAF6)、si-TRAF6和相应的对照(miR-NC mimic、pcDNA3.1-NC、si-NC)转染细胞,实验分为对照组(control组)、SP组、SP+miR-NC组、SP+miR-124-3p mimic组、SP+miR-124-3p mimic+pc-NC组、SP+miR-124-3p mimic+pc-TRAF6组。除control组外,其他各组将SP以1∶30的感染复数(MOI)感染细胞。感染24h后,逆转录定量聚合酶链式反应(RT-qPCR)检测细胞中miR-124-3p、TRAF6 mRNA水平;流式细胞术检测细胞凋亡;ELISA法检测细胞培养上清液中白细胞介素(IL)-1β、肿瘤坏死因子α(TNF-α)和转化生长因子-β1(TGF-β1)水平;免疫荧光染色检测核因子-κB p65(NF-κB p65)表达;蛋白质印迹(Western blot)检测细胞中凋亡相关标志物Caspase-3、Bcl-2、Bax和TRAF6、NFκB p65蛋白表达。结果SP感染可降低A549细胞中miR-124-3p水平,升高TRAF6、TNF-α、IL-1β和TGF-β1水平,促进NFκB p65的核转位,同时升高Caspase-3、Bax蛋白水平,降低Bcl-2蛋白水平,诱导细胞凋亡(均P<0.05);过表达miR124-3p可降低TRAF6、TNF-α、IL-1β和TGF-β1水平以及Caspase-3、Bax蛋白表达,升高Bcl-2蛋白表达,抑制NF-κB p65的核转位,减少细胞凋亡(均P<0.05);在miR-124-3p过表达的基础上,上调TRAF6表达可促进NF-κB p65核转位,增强细胞凋亡,阻断miR-124-3p mimic对细胞凋亡的保护作用。结论过表达miR-124-3p可能通过靶向下调TRAF6,抑制NF-κB核转位,减少SP感染诱导的AEC凋亡。
Objective To investigate the molecular mechanism of MicroRNA-124-3p(miR-124-3p)in the apoptosis of alveolar epithelial cells(AEC)induced by Streptococcus pneumoniae(SP)infection.Methods A549 cells were cultured in vitro,and transfected with miR-124-3p mimic,pcDNA3.1-tumor necrosis factor receptor-associated factor 6(TRAF6),si-TRAF6 and corresponding controls(miR-NC mimic,pcDNA3.1-NC,si-NC),respectively,then they were separated into control group,SP group,SP+miR-NC group,SP+miR-124-3p mimic group,SP+miR-124-3p mimic+pc-NC group,and SP+miR-124-3p mimic+pc-TRAF6 group.Except for the control group,the cells in other groups infected with SP at a multiplicity of infection(MOI)of 1∶30.24 hours after infection,reverse transcription quantitative polymerase chain reaction(RT-qPCR)was performed to measure the levels of miR-124-3p and TRAF6 mRNA in cells;flow cytometry was performed to measure apoptosis;ELISA was performed to measure the levels of interleukin(IL)-1β,tumor necrosis factorα(TNF-α)and transforming growth factor-β1(TGF-β1)in cell culture supernatants;immunofluorescence staining was performed to measure nuclear factor-κB p65(NF-κB p65)expression;Western blot was performed to measure the protein expression of apoptosis-related markers Caspase-3,Bcl-2,Bax and TRAF6,NF-κB p65 in cells.Results SP infection was able to reduce the level of miR-124-3p in A549 cells,increase the levels of TRAF6,TNF-α,IL-1βand TGF-β1,promote the nuclear translocation of NF-κB p65,increase the protein levels of Caspase-3 and Bax,decrease the protein level of Bcl-2,and induce apoptosis(all P<0.05);overexpression of miR-124-3p was able to reduce the levels of TRAF6,TNF-α,IL-1βand TGF-β1,the protein expression of Caspase-3 and Bax,increase the protein expression of Bcl-2,inhibit the nuclear translocation of NF-κB p65,and reduce apoptosis(all P<0.05);on the basis of miR-124-3p overexpression,up-regulation of TRAF6 expression was able to promote NF-κB p65 nuclear translocation,enhance apoptosis,and block the protective effect of miR-124-3p mimic on apoptosis.Conclusion Overexpression of miR-124-3p may reduce SP infection-induced AEC apoptosis by targeting down-regulation of TRAF6 and inhibiting NF-κB nuclear translocation.
作者
李娟
李晶晶
Li Juan;Li Jingjing(The Central Hospital of Yongzhou,Yongzhou Hospital Affiliated to University of South China,Yongzhou,Hunan 425100,China;Yueyang People’s Hospital,Yueyang,Hunan 414000,China)
出处
《首都食品与医药》
2025年第14期32-37,共6页
Capital Food Medicine
