流感病毒诱导的小鼠肺上皮细胞损伤及外泌体miRNA差异表达谱研究

Influenza virus-induced lung epithelial cell injury and differential expression profile of miRNAs in exosomes

目的:通过研究甲型流感病毒刺激后小鼠肺上皮细胞源性外泌体的微小RNA(miRNA)差异表达谱,探讨流感病毒诱导肺上皮细胞损伤的机制。方法:采用甲型流感病毒(MOI=0.2)诱导小鼠肺上皮MLE-12细胞,同时设空白对照组,通过CCK-8法检测不同诱导时间下的细胞活力,明确甲型流感病毒诱导细胞损伤的最佳条件。通过流式细胞术检测细胞凋亡水平,RT-qPCR和Western blot检测炎症反应相关因子的表达水平。采用超速离心法提取甲型流感病毒诱导的MLE-12细胞上清液中的外泌体,并通过透射电镜、纳米颗粒跟踪分析、Western blot及高通量单细胞测序检测外泌体形态、粒径、浓度、膜标志蛋白表达及其miRNA差异表达谱。结果:CCK-8实验结果显示,MOI=0.2的甲型流感病毒干预8 h的MLE-12细胞活力抑制率最为符合本研究需要。以该条件干预细胞,凋亡率(24.11%±0.17%)显著升高;甲型流感病毒核蛋白(NP)、肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的mRNA和蛋白表达水平均显著升高。透射电镜观察到提取的外泌体为圆形的囊泡状;纳米颗粒跟踪分析检测到甲型流感病毒感染的细胞上清液中外泌体的粒径分布发生明显改变;Western blot检测到外泌体膜标志蛋白(CD63、CD81和TSG101)的表达。细胞测序结果共检测出154个差异表达的miRNAs,其中82个呈上调趋势,72个呈下调趋势;挑选出10个进行RT-qPCR验证,有6个与测序结果一致。结论:流感病毒诱导小鼠肺上皮细胞损伤的机制可能与外泌体特异性miRNA有关。

AIM:To explore the mechanism of influenza virus-induced lung epithelial injury by studying microRNA(miRNA)expression profiles of lung epithelial cell-derived exosomes induced by influenza A virus.METHODS:Mouse lung epithelial MLE-12 cells were infected by influenza A virus(MOI=0. 2),and a blank control group was set simultaneously. In order to determine the optimal intervention conditions for influenza A virus-induced cell injury,the cell viability at different time pionts was detected by Cell Counting Kit-8. The cell apoptosis was detected by flow cytometry,and the mRNA expression levels of inflammatory response-related factors were detected by RT-qPCR. Exosomes in the supernatant of MLE-12 cells induced by influenza A virus were extracted by hypervelocity centrifugation method,and their morphological change,particle size,concentration,membrane marker proteins and miRNA differential expression profile were detected by electron microscopy,nanoparticle tracking analysis(NTA),Western blot and highthroughput single-cell sequencing.RESULTS:After exposure to influenza A virus(MOI=0. 2)for 8 h,increased apoptosis rate(24. 11%±0. 17%)of the MLE-12 cells and up-regulated mRNA and protein expression levels of inflammatory response-related factors such as influenza A virus nucleoprotein(NP),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were observed,suggesting that influenza virus can induce apoptosis and aggravate cell damage by activating lung epithelial cell inflammatory response. The exosomes showed round vesicles under electron microscope,and NTA results showed that the particle size distribution of the exosomes significantly changed in culture medium of influenza A virus-infected cells. Positive expression of the membrane marker proteins(CD63,CD81 and TSG101)was measured by Western blot. A total of154 differentially expressed miRNAs were detected by cell sequencing,82 of which were up-regulated,while 72 were decreased. Ten miRNAs were selected for RT-qPCR,and the results of 6 of them were consistent with the sequencing results.CONCLUSION:The mechanism of influenza virus-induced lung epithelial cell injury in mice may be related to the changes of specific miRNAs in exosomes.