脂多糖诱导人肺血管内皮细胞骨架重构及相关miRNA谱分析  

LPS-induced endothelial cytoskeletonremodeling in human lung vessels and related miRNAs-profiling

作　　者：吕玉珍 喻文琴 杨宇璐 薛小兰 马海滨[2] 马晓薇[3] LYU Yuzhen;YU Wenqin;YANG Yulu;XUE Xiaolan;MA Haibin;MA Xiaowe(Clinical Medical College,Ningxia Medical University,Yinchuan 750004;Human Stem Cell Institute,General Hospital of Ningxia Medical University,Yinchuan 750004;Intensive Care Unit,Cardiocerebral Vascular Disease Hospital,General Hospital of Ningxia Medical University,Yinchuan 750012,China)

机构地区：[1]宁夏医科大学临床医学院,宁夏银川750004 [2]宁夏医科大学总医院干细胞研究所,宁夏银川750004 [3]宁夏医科大学总医院心脑血管病ICU,宁夏银川750012

出　　处：《细胞与分子免疫学杂志》2023年第7期592-598,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81860350);宁夏自然科学基金(2020AAC02002)。

摘　　要：目的 探讨脂多糖(LPS)对人肺血管内皮细胞(HPVEC)骨架的影响及相关差异表达微小RNA(miRNA)谱的生物学分析。方法 通过显微镜观察HPVEC形态,异硫氰酸荧光素标记的鬼笔环肽(FITC-phalloidin)染色实验观察细胞骨架、免疫荧光细胞化学染色检测血管内皮钙黏蛋白(VE-cadherin)表达,小管生成实验检测血管生成能力,细胞迁移实验检测细胞迁移能力,JC-1线粒体膜电位检测确定细胞凋亡情况。采用高通量测序检测miRNA表达的变化,筛选出差异表达显著的miRNA,利用预测miRNA靶标基因的软件miRanda和预测miRNA结合位点的软件TargetScan进行靶基因预测,用基因本体论(GO)数据库和京都基因和基因组百科全书(KEGG)数据库对靶基因进行功能富集,进一步对与HPVEC损伤明显相关的miRNA进行生物学分析。结果 LPS诱导HPVEC损伤后形态变圆,细胞骨架完整性破坏,VE-cadherin表达下降,血管形成及迁移能力下降,凋亡增加,测序结果发现差异miRNA共229个,其中上调表达84个,下调表达145个,对这些差异miRNA进行靶基因预测及功能富集分析发现其主要富集于细胞连接及骨架调节、细胞黏附过程、炎症相关通路等通路。结论 在ALI体外模型中,多个miRNA协同参与HPVEC骨架重构、屏障功能降低、血管生成、迁移及凋亡等过程。Objective To investigate the effects of lipopolysaccharide(LPS)on human pulmonary vascular endothelial cells(HPVECs)cytoskeleton and perform biological analysis of the microRNA(miRNA)spectrum.Methods The morphology of HPVECs was observed by microscope,the cytoskeleton by FITC-phalloidin staining,and the expression of VE-cadherin was detected by immunofluorescence cytochemical staining;the tube formation assay was conducted to examine the angiogenesis,along with cell migration test to detect the migration,and JC-1 mitochondrial membrane potential to detect the apoptosis.Illumina small-RNA sequencing was used to identify differentially expressed miRNAs in NC and LPS group.The target genes of diferentially expressed miRNAs were predicted by miRanda and TargetScan,and the functional and pathway enrichment analysis was performed on Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Further biological analysis of related miRNAs was carried out.Results After the LPS got induced,the cells became round and the integrity of cytoskeleton was destroyed.The decreased expression of VE-cadherin was also observed,along with the decreased ability of angiogenesis and migration,and increased apoptosis.Sequencing results showed a total of 229 differential miRNAs,of which 84 miRNA were up-regulated and 145 miRNA were down-regulated.The target gene prediction and functional enrichment analysis of these differential miRNA showed that they were mainly concentrated in pathways related to cell connection and cytoskeleton regulation,cell adhesion process and inflammation.Conclusion In vitro model of lung injury,multiple miRNAs are involved in the process of HPVECs cytoskeleton remodeling,the reduction of barrier function,angiogenesis,migration and apoptosis.

关 键 词：人肺血管内皮细胞(HPVEC) 脂多糖(LPS) 屏障功能 微小RNA(miRNA) 

分 类 号：R563[医药卫生—呼吸系统] R392-33[医药卫生—内科学] Q731[医药卫生—临床医学] R965[生物学—分子生物学]