急性臭氧暴露对大鼠肺泡灌洗液中外泌体miRNA表达谱的影响

Effect of acute ozone exposure on miRNA expression profile of exosomes in alveolar lavage fluid of rats

目的明确急性臭氧暴露后大鼠肺泡灌洗液来源的外泌体中微小RNA(microRNA,miRNA)表达谱的变化,并分析其与肺损伤的潜在关系。方法将成年雄性Wistar大鼠放入气体染毒舱内分别暴露于0、0.5和2 ppm臭氧6 h。实验结束后24 h麻醉大鼠,收集肺泡灌洗液并提取外泌体。采用miRNA-seq技术检测外泌体中差异表达的miRNA,使用miRDB数据库对组间差异表达Top 10 miRNA进行靶基因预测,并对靶基因进行Gene Ontology功能富集和KEGG Pathway分析。利用qRT-PCR方法对表达丰度较高且组内均一性较好的差异表达miRNA进行验证。结果与0 ppm组相比,0.5 ppm组大鼠肺泡灌洗液中外泌体miRNA有8个上调、55个下调,而2 ppm组中有32个上调、61个下调。与0.5 ppm组相比,2 ppm组大鼠肺泡灌洗液中外泌体miRNA有90个上调、74个下调。差异表达Top 10 miRNA的靶基因主要参与调控DNA结合、杂环化合物结合和影响转录等方面,并与癌症、MAPK、PIK3-AKT、TNF、ErbB、线粒体自噬和FoxO等信号通路密切相关。qRT-PCR结果表明测序结果基本可靠。结论急性臭氧暴露可导致大鼠肺泡灌洗液中外泌体miRNA表达谱发生变化,差异miRNA可能参与了臭氧暴露所致的肺组织炎症、肺损伤等病理进程。

Objective To clarify the changes in the microRNA(miRNA)expression profile of rat alveolar lavage-fluid-derived exosomes after acute ozone exposure and analyze its potential relationship with lung injury.Methods Adult male Wistar rats were exposed to 0 ppm,0.5 ppm,and 2 ppm ozone for 6 h in a gas exposure chamber.After 24 h,the rats were anesthetized,the alveolar lavage fluid was collected,and exosomes were extracted.Differentially expressed miRNAs in exosomes were detected by miRNA-seq technology.miRDB databases were used to predict the target genes of the top 10 differentially expressed miRNAs among the groups,and Gene Ontology function enrichment and KEGG Pathway analyses were performed on the target genes.The differentially expressed miRNAs with high expression abundance and good uniformity were verified by quantitative reverse-transcription PCR(qRT-PCR).Results Compared with the findings in the 0 ppm group,there were 8 up-regulated and 55 down-regulated miRNAs in the 0.5 ppm group,and 32 up-regulated and 61 down-regulated miRNAs in the 2 ppm group.Compared with the findings in the 0.5 ppm group,90 exosomal miRNAs were up-regulated and 74 were down-regulated in the 2 ppm group.The target genes of top 10 differentially expressed miRNAs were mainly involved in regulating DNA binding,heterocyclic compound binding,and affecting transcription,and were closely related to cancer,MAPK,PIK3-AKT,TNF,ErbB,mitophagy,and FoxO signaling pathways.The qRT-PCR showed that the sequencing result were basically reliable.Conclusions Acute ozone exposure can lead to changes in the expression profile of exosomal miRNA in the alveolar lavage fluid of rats.Differentially expressed miRNAs may be involved in pathological processes,such as lung tissue inflammation and lung injury,caused by ozone exposure.