Comparative transcriptomic analysis of vascular endothelial cells after hypoxia/re-oxygenation induction based on microarray technology  

基于微阵列技术的缺氧/复氧诱导下血管内皮细胞转录组分析

作　　者：Jia XU Jiu-kun JIANG Xiao-lin LI Xiao-peng YU Ying-ge XU Yuan-qiang LU 

机构地区：[1]Department of Emergency Medicine,the First Affiliated Hospital,School of Medicine,Zhejiang University,Hangzhou 310003,China [2]Department of Geriatric Medicine,the First Affiliated Hospital,School of Medicine,Zhejiang University,Hangzhou 310003,China [3]Zhejiang Provincial Key Laboratory for Diagnosis and Treatment of Aging and Physic-chemical Injury Diseases,the First Affiliated Hospital,School of Medicine,Zhejiang University,Hangzhou 310003,China [4]State Key Laboratory for Diagnosis and Treatment of Infectious Diseases,the First Affiliated Hospital,School of Medicine,Zhejiang University,Hangzhou 310003,China

出　　处：《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》2020年第4期291-304,共14页浙江大学学报（英文版）B辑（生物医学与生物技术）

基　　金：Project supported by the National Natural Science Foundation of China(Nos.81801572 and 81272075);the Foundation of Key Discipline Construction of Zhejiang Province for Traditional Chinese Medicine(No.2017-XKA36);the Foundation of Key Research Project of Zhejiang Province for Traditional Chinese Medicine(No.2019ZZ014);the Medical and Health Science Foundation of Zhejiang Province(No.2019327552);the Key Research and Development Program of Zhejiang Province(No.2019C03076);the General Research Program of Zhejiang Provincial Department of Medical and Health(No.2013KYA066);the Opening Foundation of State Key Laboratory for the Diagnosis and Treatment of Infectious Diseases(Nos.2018KF02 and 2019KF06);the Program of Education Department of Zhejiang Province(No.Y201738150),China。

摘　　要：Objective: To provide comprehensive data to understand mechanisms of vascular endothelial cell(VEC) response to hypoxia/re-oxygenation. Methods: Human umbilical vein endothelial cells(HUVECs) were employed to construct hypoxia/re-oxygenation-induced VEC transcriptome profiling. Cells incubated under 5% O2, 5% CO2, and 90% N2 for 3 h followed by 95% air and 5% CO2 for 1 h were used in the hypoxia/re-oxygenation group. Those incubated only under 95% air and 5% CO2 were used in the normoxia control group. Results: By using a well-established microarray chip consisting of 58 339 probes, the study identified 372 differentially expressed genes. While part of the genes are known to be VEC hypoxia/re-oxygenation-related, serving as a good control, a large number of genes related to VEC hypoxia/re-oxygenation were identified for the first time. Through bioinformatic analysis of these genes, we identified that multiple pathways were involved in the reaction. Subsequently, we applied real-time polymerase chain reaction(PCR) and western blot techniques to validate the microarray data. It was found that the expression of apoptosis-related proteins, like pleckstrin homology-like domain family A member 1(PHLDA1), was also consistently up-regulated in the hypoxia/re-oxygenation group. STRING analysis found that significantly differentially expressed genes SLC38A3, SLC5A5, Lnc-SLC36 A4-1, and Lnc-PLEKHJ1-1 may have physical or/and functional protein–protein interactions with PHLDA1. Conclusions: The data from this study have built a foundation to develop many hypotheses to further explore the hypoxia/re-oxygenation mechanisms, an area with great clinical significance for multiple diseases.目的:应用全转录组芯片研究缺氧/复氧诱导下人脐静脉内皮细胞(HUVEC)的转录组轮廓。创新点:血管内皮细胞(VEC)缺氧/复氧损伤被视定为许多生理和病理过程中导致器官功能障碍的重要驱动因素。然而,其详细病理生理机制和基因表达谱信息尚未阐明。本研究首次应用全转录组芯片技术研究VEC缺氧/复氧诱导下的转录组轮廓。方法:采用缺氧孵育3h后复氧1h的HUVEC为缺氧/复氧组,同时常氧孵育的HUVEC为常氧对照组。应用含58 339条探针的全转录组芯片检测每组三个样本。对差异表达基因进行生信分析和功能验证。结论:本研究发现372个有意义的差异表达基因探针。相关基因涵盖多种途径和功能,例如氧自由基的产生、钙超载、炎症、糖脂代谢、内皮细胞增殖、分化、细胞骨架及通透性调节、细胞裂解、凋亡和血管生成。另外,实验进一步表明,差异表达基因pleckstrin同源样域家族A成员1(PHLDA1)的m RNA和蛋白质表达结果与微阵列结果一致。STRING分析发现,PHLDA1可能与差异表达基因SLC38A3、SLC5A5、Lnc-SLC36A4-1和Lnc-PLEKHJ1-1具有物理性和/或功能性相互作用,这有望揭示VEC在缺氧/复氧环境下长链非编码RNA(lnc RNA)的相关机制。

关 键 词：Human umbilical vein endothelial cells(HUVECs) HYPOXIA Re-oxygenation MICROARRAY Pleckstrin homology-like domain family A member 1(PHLDA1) Long non-coding RNA(lncRNA) 

分 类 号：R592[医药卫生—老年医学]