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王艳琼,董利利,李 敏,张 磊,徐沙沙,汤 昱.miR-27a 调控 PI3K/ AKT / mTOR 通路介导的自噬对肺炎链球菌诱导人肺泡上皮细胞损伤的影响[J].中国比较医学杂志,2021,31(11):27~34.
miR-27a 调控 PI3K/ AKT / mTOR 通路介导的自噬对肺炎链球菌诱导人肺泡上皮细胞损伤的影响
Effect of miR-27a on Streptococcus pneumoniae (SP)-induced injury of human alveolar epithelial cells by regulating autophagy mediated by the PI3K / AKT/ mTOR pathway
投稿时间:2020-11-27  
DOI:10. 3969 / j.issn.1671-7856. 2021. 11. 005
中文关键词:  miR-27a  PI3K/ AKT/ mTOR  自噬  肺炎链球菌  肺泡上皮细胞  损伤
英文关键词:miR-27a  PI3K/ AKT/ mTOR  autophagy  Streptococcus pneumoniae  alveolar epithelial cells  injury
基金项目:
作者单位E-mail
王艳琼 郑州大学附属儿童医院 河南省儿童医院 郑州儿童医院呼吸科,郑州 450018 yanqiongwork87@ 163.com 
董利利 郑州大学附属儿童医院 河南省儿童医院 郑州儿童医院呼吸科,郑州 450018  
李 敏 郑州大学附属儿童医院 河南省儿童医院 郑州儿童医院呼吸科,郑州 450018  
张 磊 郑州大学附属儿童医院 河南省儿童医院 郑州儿童医院呼吸科,郑州 450018  
徐沙沙 郑州大学附属儿童医院 河南省儿童医院 郑州儿童医院呼吸科,郑州 450018  
汤 昱 郑州大学附属儿童医院 河南省儿童医院 郑州儿童医院呼吸科,郑州 450018 tangyu010312@ sina.com 
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中文摘要:
       目的 探讨 miR-27a 调控 PI3K/ AKT/ mTOR 通路介导的自噬对肺炎链球菌(SP)诱导人肺泡上皮细胞(HPAEpiC)损伤的影响。 方法 用 1× 108 CFU/ mL SP 诱导 HPAEpiC,在诱导前 48 h 使用 Lipofectamine 3000 转染试剂盒分别转染细胞 miR-27a-NC(miR-27a-NC 组)、miR-27a-inhibitor(miR-27a-inhibitor 组)、pcDNA-NC( pcDNA- NC 组)、pcDNA-PI3K ( pcDNA-PI3K 组), 共转染细胞 miR-27a-NC 和 Si-NC ( miR-27a-NC + Si-NC 组)、 miR-27a- inhibitor 和 Si-NC(miR-27a-inhibitor+Si-NC 组)、miR-27a-NC 和 Si-PI3K(miR-27a-NC+Si-PI3K 组)、miR-27a-inhibitor 和 Si-PI3K(miR-27a-inhibitor+Si-PI3K 组)。另取非诱导细胞为对照组,诱导且未转染细胞为诱导组。 qRT-PCR 检测正常 HPAEpiC 和 SP 诱导的 HPAEpiC 中 miR-27a 表达水平;生物信息学预测和双荧光素酶验证 miR-27a 和 PI3K 靶向关系;CCK-8 法检测细胞增殖活性;流式细胞仪检测细胞凋亡率;ELISA 法检测细胞上清液中 IL-6 和 IL-10 含 量;Western blot 检测细胞中 PI3K、Beclin1 蛋白表达水平、LC3-II/ I 比值以及 AKT、mTOR 蛋白磷酸化水平。 结果 与对照组相比,诱导组细胞 miR-27a 表达水平、细胞凋亡率、IL-6 含量、Beclin1 蛋白表达水平和 LC3-II/ I 比值升高 (P<0. 05),PI3K 蛋白表达水平、细胞增殖率、IL-10 含量、AKT、mTOR 蛋白磷酸化水平降低(P<0. 05)。与 miR-27a- NC 组相比,miR-27a-inhibitor 组细胞 miR-27a 表达水平、细胞凋亡率、IL-6 含量、Beclin1 蛋白表达水平和 LC3-II/ I 比值降低(P<0. 05),细胞增殖率、IL-10 含量、AKT、mTOR 蛋白磷酸化水平升高(P<0. 05)。 与 pcDNA-NC 组相比, pcDNA-PI3K 组细胞凋亡率、IL-6 含量、Beclin1 蛋白表达水平和 LC3-II/ I 比值降低(P<0. 05),PI3K 蛋白表达水平、细胞增殖率、IL-10 含量、AKT、mTOR 蛋白磷酸化水平升高(P<0. 05)。 结论 在 SP 诱导 HPAEpiC 损伤过程中, miR-27a 能够靶向抑制 PI3K/ AKT/ mTOR 信号通路,促进自噬,而抑制 miR-27a 可激活 PI3K/ AKT/ mTOR 信号通路, 抑制自噬,缓解 SP 所致 HPAEpiC 损伤。
英文摘要:
       Objective To investigate the effect of miR-27a on Streptococcus pneumoniae ( SP) -induced injury of HPAEpiCs by regulating autophagy mediated by the PI3K/ AKT / mTOR pathway. Methods HPAEpiCs were infected with 1× 108 CFU/ mL SP. At 48 hours before induction the cells were transfected with miR-27a-NC ( miR-27a-NC group) , miR-27a-inhibitor ( miR-27a-inhibitor group) , pcDNA-NC ( pcDNA-NC group) or pcDNA-PI3K ( pcDNA- PI3K group) or cotransfected with miR-27a-NC and Si-NC (miR-27a-NC+Si-NC group) , miR-27a inhibitor and Si-NC (miR-27a inhibitor+Si-NC group) , miR-27a-NC and Si-PI3K (miR-27a-NC+Si-PI3K group) , or miR-27a-inhibitor and Si-PI3K (miR-27a-inhibitor+Si-PI3K group) using a Lipofectamine 3000 transfection kit. Non-induced cells were used as the control group, and induced, but untransfected, cells were used as the induction group. qRT-PCR was used to measure the expression level of miR-27a in normal and SP-induced alveolar epithelial cells. Bioinformatics prediction and dual luciferase assays were used to verify the targeting relationship between miR-27a and PI3K. CCK-8 Assays was used to assess proliferation activity. Flow cytometry was used to measure the apoptosis rate. ELISA were used to detect the contents of IL-6 and IL-10 in culture supernatants. Western blot was used to measure the expression levels of PI3K and Beclin1, the LC3-II/ I ratio and the phosphorylation levels of AKT and mTOR. Results Compared with the control group, the expression level of miR-27a, apoptosis rate, IL-6 content, Beclin1 protein expression level and LC3-II/ I ratio were higher in the induction group (P<0. 05) and the PI3K protein expression level, cell proliferation rate, IL-10 content and phosphorylation levels of AKT and mTOR were lower (P<0. 05) . Compared with the miR-27a-NC group, the expression level of miR-27a, apoptosis rate, IL-6, Beclin1 protein expression level and LC3-II/ I ratio were lower in the miR-27a-inhibitor group content ( P< 0. 05) , the cell proliferation rate, content of IL-10, the protein phosphorylation levels of AKT and mTOR were higher ( P< 0. 05) . Compared with the pcDNA-NC group, the apoptosis rate, IL-6 content, Beclin1 protein expression level and LC3-II/ I ratio were lower in the pcDNA-PI3K group ( P< 0. 05) and the PI3K protein expression level, cell proliferation rate, IL-10 content and phosphorylation levels of AKT and mTOR were higher (P<0. 05) . Conclusions During injury of HPAEpiCs induced by SP, miR-27a inhibits the PI3K/ AKT / mTOR signaling pathway and promotes autophagy. Inhibition of miR-27a activates the PI3K/ AKT / mTOR signaling pathway, inhibits autophagy and alleviates SP-induced injury of HPAEpiCs.
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