低氧诱导视网膜色素上皮细胞损伤的生物学机制转录组测序分析  

Transcriptomics analysis of hypoxia-induced retinal pigment epithelium cell injury

作　　者：卢聪 史平玲 杨琪翔 宋昊 李苗[1] 张贝贝[1] 宋宗明 Lu Cong;Shi Pingling;Yang Qixiang;Song Hao;Li Miao;Zhang Beibei;Song Zongming(Henan University People's Hospital,Department of Ophthalmology,Henan Provincial People's Hospital,Henan Eye Hospital,Henan Eye Institute,Zhengzhou 450003,China)

机构地区：[1]河南大学人民医院,河南省人民医院眼科,河南省立眼科医院,河南省眼科研究所,郑州450003

出　　处：《中华实验眼科杂志》2021年第6期505-514,共10页Chinese Journal Of Experimental Ophthalmology

基　　金：河南省科技攻关项目(192102310075);河南省医学科技攻关省部共建项目(SBGJ2018080);河南省卫生健康科技英才海外研修工程项目(HWYX2019131)。

摘　　要：目的利用转录组测序(RNA-seq)和生物信息学技术对低氧与常氧条件下人视网膜色素上皮细胞系ARPE-19细胞的差异表达基因(DEGs)及信号通路的变化进行分析,探讨低氧诱导ARPE-19细胞损伤的生物学机制。方法将ARPE-19细胞分为低氧处理组和常氧对照组,分别采用体积分数1%和21%O 2处理细胞8、24、48、72 h,采用实时荧光定量PCR法检测不同时间点血管内皮生长因子(VEGF)和低氧诱导因子(HIF-1α)mRNA相对表达量。分别对2个组处理后8 h和24 h的ARPE-19细胞进行RNA-seq及生物信息学分析,以|log 2 FC|≥1且P≤0.05为条件筛选出DEGs,并对DEGs进行聚类热图分析、基因本体论(GO)功能富集分析、京都基因与基因组百科全书(KEGG)通路富集分析及蛋白-蛋白互作(PPI)网络分析。采用实时荧光定量PCR法检测低氧处理ARPE-19细胞24 h后DEGs中可能与低氧有关的基因mRNA相对表达量。采用细胞活力检测试剂盒验证2个组不同时间点低氧对ARPE-19细胞的损伤作用。结果低氧处理组8、24、48、72 h VEGF和低氧处理组8、24、48 h HIF-1αmRNA相对表达量均高于常氧对照组,差异均有统计学意义(均P<0.05),其中低氧处理后8 h和24 h各因子mRNA表达差异较大。低氧诱导8 h,低氧处理组与常氧对照组间筛选显著DEGs共62个,其中显著上调基因45个,显著下调基因17个;低氧诱导24 h,2个组间筛选显著DEGs共255个,其中显著上调基因228个,显著下调基因27个。DEGs的GO功能分析主要富集在蛋白质降解、核苷酸生物合成及物质转运等进程。KEGG通路分析主要富集在PI3K-Akt、cGMP-PKG以及其他与代谢、细胞周期、细胞生长和细胞凋亡密切相关的信号通路。PPI网络分析发现核心基因HPCA、MT3和NOS3等。实时荧光定量PCR检测结果示,低氧处理ARPE-19细胞24 h后,低氧相关基因DEPP1、NPPB、PDZK1、HILPDA、TCEA3、NDRG1和RORC的mRNA表达水平升高,TFRC、NQO1的mRNA表达水平降低,差异Objective To analyze differentially expressed genes(DEGs)and the changes of signal pathways in human retinal pigment epithelium cells(ARPE-19)under hypoxic and normoxic conditions and to explore the biological mechanism of hypoxia-induced ARPE-19 cell damage via transcriptome sequencing(RNA-seq)and bioinformatics technology.Methods The ARPE-19 cells were divided into the hypoxia treatment group and the normoxia control group treated with 1%and 21%O 2 by volume for 8,24,48,72 hours,respectively.The relative expression levels of vascular endothelial growth factor(VEGF)and hypoxia-inducible factor-1α(HIF-1α)mRNA were detected with real-time fluorescent quantitative PCR at different time points.RNA-seq and bioinformatics analysis were performed at 8 hours and 24 hours after hypoxia and normoxia treatment.DEGs were screened out under the conditions of|log 2FC|≥1 and P≤0.05.Then the cluster heat map analysis,Gene Ontology(GO)functional enrichment analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis and protein-protein interaction network analysis were also carried out.Real-time fluorescent quantitative PCR was employed at 24 hours after hypoxia to detect the relative mRNA expression of genes that might be related to hypoxia in DEGs.Cell viability kit was used to verify and compare the damage effect of hypoxia on ARPE-19 cells at different time points between the two groups.Results The relative mRNA expression levels of VEGF at 8,24,48 and 72 hours after hypoxia treatment and the relative HIF-1αmRNA expression levels at 8,24 and 48 hours after hypoxia treatment were significantly higher than those of the normoxia control group(all at P<0.05).There were large differences in the mRNA expression levels at 8-hour and 24-hour treatment between the two groups.A total of 62 significant DEGs were screened between the hypoxia treatment group and the normoxia control group after 8-hour hypoxia treatment,among which 45 genes were significantly up-regulated and 17 genes were significantly down-re

关 键 词：视网膜色素上皮细胞 低氧 差异表达基因 转录组测序 

分 类 号：R774.1[医药卫生—眼科]