中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (12): 2104-2111.doi: 10.4103/1673-5374.262589

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

氧糖剥夺恢复后损伤脑微血管内皮细胞中可见多种新型差异表达的环状RNA

  

  • 出版日期:2019-12-15 发布日期:2019-12-15
  • 基金资助:

    国家自然科学基金项目青年项目（81601058），重庆市渝中区基础研究前沿科学探索基金项目（20180106）

Novel circular RNAs expressed in brain microvascular endothelial cells after oxygen-glucose deprivation/recovery

Wei Liu 1 , Chao Jia 2 , Li Luo 3, 4 , Hai-Lian Wang 1 , Xiao-Li Min 5 , Jiang-Hui Xu 1 , Li-Qing Ma 1 , Xia-Min Yang 1 , Ying-Wei Wang 1 , Fei-Fei Shang 3   

  1. 1 Department of Anesthesiology, Huashan Hospital, Fudan University, Shanghai, China
    2 Department of Medical Ultrasound, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    3 Institute of Life Sciences, Chongqing Medical University, Chongqing, China
    4 Chongqing Foreign Language School, Chongqing, China
    5 Department of Cerebrovascular Diseases, The Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan Province, China
  • Online:2019-12-15 Published:2019-12-15
  • Contact: Ying-Wei Wang, MD, wangyingwei@yahoo.com; Fei-Fei Shang, PhD, sff_phoenix@cqmu.edu.cn.
  • Supported by:

    This work was supported by the National Natural Science Foundation for Young Scientists of China, No. 81601058 (to WL); and Basic Research and Frontier Science Exploration Foundation of Yuzhong District, Chongqing, China, No. 20180106 (to FFS).

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摘要：

环状RNA通过基因头对尾剪接产生，且在所有多细胞生物中普遍表达，其生物学功能已被越来越多地关注。脑缺血再灌注损伤诱导的脑微血管内皮细胞功能障碍是血脑屏障破坏的基础。但是脑微血管内皮细胞中环状RNA的表达谱和潜在功能尚不清楚。实验首先提取大鼠脑微血管内皮细胞，以无糖培养基95％N2，5％CO2，37℃培养4h后，再以完全培养基培养6h。提取细胞中的RNA，使用Find_circ和CIRI2软件鉴定环状RNA，通过DAVID Functional Annotation Tool对DE环状RNA宿主基因和差异表达的mRNA的交叉基因进行功能和途径富集分析，以Miranda软件用于预测可能被circRNA吸收的miRNA，以Cytoscape用于描述环状RNA-miRNA相互作用网络。结果显示正常和氧糖剥夺/恢复的脑微血管内皮细胞中共存在1288个环状RNA。氧糖剥夺/恢复的脑微血管内皮细胞中有211个差异表达的circRNA上调，326个差异表达的circRNA下调。这些差异表达的circRNA的宿主基因与DE mRNA的宿主基因重叠。功能富集分析进一步研究共有基因，发现circRNA可能有助于钙离子功能和环鸟苷3'，5'-单磷酸环磷酸鸟苷-环磷酸鸟苷依赖性蛋白激酶信号传导通路。实时定量PCR测定从重叠宿主基因转录的环状RNA的水平，其中最高倍数变化值的10个环状RNA中的8个被成功验证。随后通过生物信息学分析探索了这8种环状RNA的环状RNA-微小RNA相互作用网络，分析鉴定了可能与DE环状RNA相互作用的微小RNA。表明这些差异表达的环状RNA在脑缺血再灌注损伤发病中是重要的，可能成为脑缺血疾病的潜在治疗靶点。实验于2018年3月22日经重庆医科大学动物委员会批准，批准号为CQMU20180086。

orcid: 0000-0003-3271-8562 (Ying-Wei Wang)
          0000-0002-2706-2100 (Fei-Fei Shang)

关键词: 环状RNAs, 脑微血管内皮细胞, 高通量测序, 氧糖剥夺/恢复, 脑缺血再灌注损伤, microRNAs, 脑缺血, 神经再生

Abstract:

Circular RNAs (circRNAs) are generated by head-to-tail splicing and are ubiquitously expressed in all multicellular organisms. Their important biological functions are increasingly recognized. Cerebral ischemia reperfusion injury-induced brain microvascular endothelial cell dysfunction is an initial stage of blood-brain barrier disruption. The expression profile and potential function of circRNAs in brain microvascular endothelial cells is unknown. Rat brain microvascular endothelial cells were extracted and cultured in glucose-free medium for 4 hours with 5% CO2 and 95% N2, and the medium was then replaced with complete growth medium for 6 hours. The RNA in these cells was then extracted. The circRNA was identified by Find_circ and CIRI2 software. Functional and pathway enrichment analysis of genes that were common to differentially expressed mRNAs and circRNA host genes was performed by the Database for Annotation, Visualization and Integrated Discovery Functional Annotation Tool. Miranda software was used to predict microRNAs that were potentially sponged by circRNAs. Furthermore, cytoscape depicted the circR-NA-microRNA interaction network. The results showed that there were 1288 circRNAs in normal and oxygen-glucose deprived/recovered primary brain microvascular endothelial cells. There are 211 upregulated and 326 downregulated differentially expressed circRNAs. The host genes of these differentially expressed circRNAs overlapped with those of differentially expressed mRNAs. The shared genes were further studied by functional enrichment analyses, which revealed that circRNAs may contribute to calcium ion function and the cyclic guanosine 3′,5′-monophosphate (CAMP) dependent protein kinase (PKα) signaling pathway. Next, quantitative reverse transcription polymerase chain reaction assays were performed to detect circRNA levels transcribed from the overlapping host genes. Eight out of the ten circRNAs with the highest fold-change identified by sequencing were successfully verified. Subsequently, the circRNA-microRNA interaction networks of these eight circRNAs were explored by bioinformatic analysis. These results demonstrate that altered circRNAs may be important in the pathogenesis of cerebral ischemia reperfusion injury and consequently may also be potential therapeutic targets for cerebral ischemia diseases. All animal experiments were approved by the Chongqing Medical University Committee on Animal Research, China (approval No. CQMU20180086) on March 22, 2018.

Key words: circRNAs, endothelial cells, RNA sequencing, cerebral ischemia reperfusion injury, microRNAs, neural regeneration