Micro-RNA与Notch信号调控肺炎克雷伯菌感染肺泡上皮细胞自噬的作用研究  

Effects of micro-RNA and Notch signaling on the regulation of autophagy in alveolar epithelial cells infected with Klebsiella pneumoniae

作　　者：师志云 尹晓丽 侯晓慧 王良方 张玉英 李刚 王文 陶佳 李莎莎 贾伟 SHI Zhi-yun;YIN Xiao-li;HOU Xiao-hui;WANG Liang-fang;ZHANG Yu-ying;LI Gang;WANG Wen;TAO Jia;LI Sha-sha;JIA Wei(Medical Experimental Center of General Hospital of Ningxia Medical University,Yinchuan,Ningxia 750004 China;Key Laboratory of Clinical Pathogenic Microorganisms of Ningxia;School of Clinical Medicine,Ningxia Medical University)

机构地区：[1]宁夏医科大学总医院医学实验中心,银川宁夏750004 [2]宁夏临床病原微生物重点实验室 [3]宁夏医科大学临床医学院

出　　处：《中国病原生物学杂志》2021年第12期1393-1397,1403,共6页Journal of Pathogen Biology

基　　金：宁夏自然科学基金项目(No.2019AAC03216,2021AAC03326);宁夏高等学校一流学科建设(宁夏医科大学国内一流建设学科临床医学)资助项目(No.NXYLXK2017A05);2019年大学生创新创业训练计划项目(No.19-2010)。

摘　　要：目的探讨Micro-RNA与Notch信号调控肺泡Ⅱ型上皮细胞自噬在肺炎克雷伯菌感染的作用机制。方法构建体外肺炎克雷伯菌(Klebsiella pneumoniae,KPN)感染人肺泡Ⅱ型上皮细胞(ACEII)A549细胞模型,荧光定量PCR筛选表达变化显著的micro-RNAs;在感染0、24、48、72 h用自噬抑制剂(3-MA)和自噬诱导剂雷帕霉素(Rapa)分别作用于A549细胞,荧光定量PCR检测micro-RNA表达的动态变化。构建A549细胞miR-144 mimics转染模型,Western blot检测Notch1的蛋白表达,显微镜观察菌落变化情况。结果与Control组相比,KPN组细胞中miR-125a、miR-144、miR-155、miR-146a、miR-9的表达水平均显著升高(均P<0.05),其中以miR-144尤为显著。随着培养时间延长,KPN组细胞中miR-144的表达水平逐渐降低;相比KPN组,KPN+3-MA组细胞中miR-144的表达水平降低更显著。成功构建miR-144 mimics转染细胞模型,与Control组相比,KPN组细胞中Notch1的蛋白表达水平显著升高(P<0.05);转染miR-144 mimics后,Notch1的蛋白表达水平显著降低(P<0.05);A549细胞在转染miR-144 mimics后感染KPN,Notch1蛋白表达水平相比KPN组显著降低(P<0.05)。菌落计数显示,24 h开始KPN+miR-144 mimics组菌落数显著高于KPN组(P<0.05),两组的菌落数均随着时间延长而增多。结论micro-RNA和Notch信号在调控肺炎克雷伯菌感染肺泡II型上皮细胞发生细胞自噬中发挥重要作用。Objective To investigate the mechanism of micro-RNA and Notch signaling in regulating the autophagy of alveolar typeⅡepithelial cells in Klebsiella pneumoniae infection.Methods An in vitro model of human alveolar typeⅡepithelial cells(ACEII A549)infected with Klebsiella pneumoniae(KPN)was constructed,and micro-RNAs with significant changes in expression were screened using fluorescence quantitative PCR.A549 cells were treated with an autophagy inhibitor(3-MA)and an autophagy inducer(rapamycin,or Rapa)0,24,48,and 72 h after infection,and dynamic changes in micro-RNA expression were determined using fluorescence quantitative PCR.A model of A549 cells transfected with miR-144 mimics was constructed,and the expression of Notch1 protein was determined using Western blotting.Colony changes were observed under a microscope.Results Compared to the control group,the levels of miR-125 a,miR-144,miR-155,miR-146 a,and miR-9 expression in the KPN group increased significantly(P<0.05 for all),and the increase in miR-144 was the most significant.With a longer duration of culturing,the level of miR-144 expression in the KPN group gradually decreased.Compared to the KPN group,the level of miR-144 expression in the KPN+3-MA group decreased significantly.A model of cells transfected with MiR-144 mimics was successfully constructed.Compared to the control group,the level of Notch1 protein expression in the KPN group increased significantly(P<0.05).After transfection with miR-144 mimics,the level of Notch1 protein expression decreased significantly(P<0.05).A549 cells were infected with KPN after transfection with miR-144 mimics,and the level of Notch1 protein expression was significantly lower than that in the KPN group(P<0.05).The colony count indicated that the number of colonies in KPN+miR-144 mimics group was significantly higher than that in the KPN group at 24 h(P<0.05),and the number of colonies in both groups increased with time.Conclusion MicroRNA and Notch signaling play an important role in regulating the autophagy of

关 键 词：肺炎克雷伯菌 肺泡Ⅱ型上皮细胞 自噬 MICRO-RNA NOTCH信号 

分 类 号：R378[医药卫生—病原生物学]