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

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1×10^(8)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。

Objective To investigate the effect of miR-27 a on Streptococcus pneumoniae(SP)-induced injury of HPAEpiCs by regulating autophagy mediated by the PI3 K/AKT/mTOR pathway. Methods HPAEpiCs were infected with 1×10^(8) CFU/mL SP. At 48 hours before induction the cells were transfected with miR-27 a-NC(miR-27 a-NC group), miR-27 a-inhibitor(miR-27 a-inhibitor group), pcDNA-NC(pcDNA-NC group) or pcDNA-PI3 K(pcDNA-PI3 K group) or cotransfected with miR-27 a-NC and Si-NC(miR-27 a-NC+Si-NC group), miR-27 a inhibitor and Si-NC(miR-27 a inhibitor+Si-NC group), miR-27 a-NC and Si-PI3 K(miR-27 a-NC+Si-PI3 K group), or miR-27 a-inhibitor and Si-PI3 K(miR-27 a-inhibitor+Si-PI3 K 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-27 a in normal and SP-induced alveolar epithelial cells. Bioinformatics prediction and dual luciferase assays were used to verify the targeting relationship between miR-27 a and PI3 K. 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 PI3 K 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-27 a, 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 PI3 K 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-27 a-NC group, the expression level of miR-27 a, apoptosis rate, IL-6, Beclin1 protein expression level and LC3-II/I ratio were lower in the miR-27 a-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-PI3 K group(P<0.05) and the PI3 K 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-27 a inhibits the PI3 K/AKT/mTOR signaling pathway and promotes autophagy. Inhibition of miR-27 a activates the PI3 K/AKT/mTOR signaling pathway, inhibits autophagy and alleviates SP-induced injury of HPAEpiCs.