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中华神经创伤外科电子杂志 ›› 2023, Vol. 09 ›› Issue (02) : 76 -84. doi: 10.3877/cma.j.issn.2095-9141.2023.02.003

临床研究

基于GEO数据库的热射病神经损伤相关基因的生物信息学分析
王蕾, 姜岱山, 朱保锋, 贾寒雨, 沈君华, 张毅†()   
  1. 226001　江苏南通，南通大学第二附属医院急诊中心
    226001　江苏南通，南通大学附属医院急诊中心
    226001　江苏南通，南通大学第二附属医院科技处
    226001　江苏南通，南通大学第二附属医院神经外科
  • 收稿日期:2022-11-04 出版日期:2023-04-15
  • 通信作者: 张毅

Bioinformatic analysis of genes related to nerve injury in heat stroke based on GEO database

Lei Wang, Daishan Jiang, Baofeng Zhu, Hanyu Jia, Junhua Shen, Yi Zhang†()   

  1. Emergency Center of the Second Affiliated Hospital of Nantong University, Nantong 226001, China
    Emergency Center of the Affiliated Hospital of Nantong University, Nantong 226001, China
    Department of Science and Technology, the Second Affiliated Hospital of Nantong University, Nantong 226001, China
    Department of Neurosurgery, the Second Affiliated Hospital of Nantong University, Nantong 226001, China
  • Received:2022-11-04 Published:2023-04-15
  • Corresponding author: Yi Zhang
  • Supported by:
    Medical Research Project of Jiangsu Provincial Health and Wellness Commission(Z2022067); Jiangsu Hospital Emergency Management Special Project(JSYGY-3-2021-JZ28); Research Project of Nantong Science and Technology Bureau(JC2021179); Nantong City Health Planning Commission Subject(MB2021018, MB2021026)
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王蕾, 姜岱山, 朱保锋, 贾寒雨, 沈君华, 张毅. 基于GEO数据库的热射病神经损伤相关基因的生物信息学分析[J]. 中华神经创伤外科电子杂志, 2023, 09(02): 76-84.

Lei Wang, Daishan Jiang, Baofeng Zhu, Hanyu Jia, Junhua Shen, Yi Zhang. Bioinformatic analysis of genes related to nerve injury in heat stroke based on GEO database[J]. Chinese Journal of Neurotraumatic Surgery(Electronic Edition), 2023, 09(02): 76-84.

目的

通过生物信息学检索热射病神经损伤的相关基因，探究热射病神经损伤的可能分子机制并进行初步验证。

方法

（1）以2022年4月作为时间截点，基于基因表达数据库（GEO）筛选热射病神经损伤相关基因表达谱数据，通过检索数据集GSE64778获得差异表达基因（DEGs），结合GeneCards和CTD数据库检索热射病相关靶基因，取交集筛选获得候选靶基因。采用R语言的"ClusterProfiler"包对热射病相关候选靶基因进行GO和KEGG富集分析；进一步使用STRING数据库构建靶基因的交互作用网络图，并对关键基因进行相关性分析获得核心基因。（2）收集江苏省南通市第一人民医院自2022年6~8月收治的热射病患者的外周血，通过PCR技术进行核心基因的表达验证。

结果

（1）GSE64778数据集的SRA数据筛选，共获得1178个DEGs，其中322个上调，856个下调。GeneCards数据库筛选出2914个基因，CTD数据库筛选出2377个基因。将GSE64778数据集差异表达排名前300的基因，与GeneCards和CTD数据库检索结果取交集，最终筛选出25个候选DEGs。（2）GO功能注释结果显示靶基因主要参与细胞凋亡、应激反应以及细胞过程的负调控，在蛋白质二聚化和蛋白结合等过程中发挥功能。KEGG通路富集分析显示候选靶基因主要富集在PI3K-Akt信号通路。PPI网络分析显示Hsp90AA1是Degree值最高的枢纽基因，且在PI3K-Akt信号通路中富集。（3）经qRT-PCR验证，Hsp90AA1在热射病早期神经损伤患者中的表达均与生物信息学分析结果一致。

结论

Hsp90AA1可能为热射病神经损伤主要的影响基因，与细胞凋亡、应激反应以及细胞过程的负调控相关。

关键词: 热射病, 神经损伤, 生物信息学, Hsp90AA1, PI3K-Akt信号通路
Objective

To search for genes related to nerve injury in heat stroke (HS) by bioinformatics, the possible molecular mechanism of nerve injury in HS was explored and preliminarily verified.

Methods

(1) Taking April 2022 as the time cut-off, the expression profile datas of nerve inject-related genes in thermal radiation disease were screened based on GEO and other databases, and differentially expressed genes (DEGs) were obtained by retrieving datas set GSE64778, combined with GeneCards and CTD databases to retrieve HS related target genes, and the candidate target genes were obtained by intersection screening. The "ClusterProfiler" package in R language was used to perform GO and KEGG enrichment analysis for HS related target genes; STRING database was further used to construct the interaction network map of target genes and correlation analysis of key genes to obtain core genes. (2) Peripheral blood of patients with HS treated in Nantong First People's Hospital of Jiangsu Province from June 2022 to August 2022 were collected, and the expression of core genes were verified by PCR technology.

Results

(1) SRA datas screening of the GSE64778 dataset yielded 1178 DEGs, of which 322 were up-regulated and 856 were down-regulated. 2914 genes were screened in the GeneCards database and 2377 genes were screened in the CTD database. The top 300 differentially expressed genes in the GSE64778 dataset were intersected with the search results of GeneCards and CTD databases, and 25 candidate DEGs were finally screened. (2) GO functional annotation results indicated that the target genes were mainly involved in apoptosis, stress response and negative regulation of cellular processes, and functioned in processes such as protein dimerization and protein binding. KEGG pathway enrichment analysis suggested that the candidate target genes were mainly enriched in PI3K-Akt signaling pathway. PPI network analysis suggested that Hsp90AA1 was the hub gene with the highest Degree value and enriched in PI3K-Akt signaling pathway. (3) qRT-PCR confirmed that the expression of Hsp90AA1 in the patients with nerve injury in the early stage of HS was consistent with the results of bioinformatics analysis.

Conclusion

Hsp90AA1 may be the main affecting gene of nerve injury in HS, which is related to apoptosis, stress response and negative regulation of cellular processes.

Key words: Heat stroke, Nerve damage, Bioinformatics, Hsp90AA1, PI3K-Akt signal pathway
表1 PCR引物序列
Tab.1 PCR primer sequence
基因 正向引物(5’-3’) 反向引物(5’-3’)
Hsp90AA1 GAAGGAATTTGAGGGGAAGACTTTA TGCCATGTAACCCATTGTTGAG
GAPDH GGAAGCTTGTCATCAATGGAAATC TGATGACCCTTTTGGCTCCC
图1 热射病发生相关候选基因的筛选A：公共数据库热射病相关数据集GSE64778中差异基因表达火山图，红点表示高表达基因，绿点表示低表达基因，灰色圆点表示没有显著差异；B：数据集GSE64778中差异表达排名前300的基因与GeneCards和CTD数据库heat stroke相关基因的交集Venn图；C：25个候选DEGs在GSE64778数据集中表达的热图；D：25个候选DEGs在GSE64778数据集中的差异表达箱线图
Fig.1 Screening of candidate genes related to the occurrence of heat emission disease
图2 热射病发生相关候选基因的功能富集分析A：BP层面GO功能分析；B：CC层面的GO功能分析；C：MF层面的GO功能分析；D：KEGG通路富集分析；不同颜色代表-log10（P value），其值越大则颜色越深，表示P值越小，差异越显著；圆圈大小表示基因富集数目，圆圈越大基因富集数目则越多
Fig.2 Functional enrichment analysis of candidate genes related to the occurrence of heat emission disease
图3 PPI分析热射病关键蛋白相互作用关系A：25个候选DEGs的PPI网络图，节点表示蛋白，边表示蛋白间的相互关联；B：25个候选DEGs按照Degree排序的条形图；C：利用数据集GSE64778分析25个候选DEGs在Heat stress组中表达量的相关性，方块颜色表示基因间相关性强弱，越红表示正相关越强，越蓝表示负相关越强，*表示相关的显著性；D：ROC曲线分析Hsp90AA1预测热射病发生的准确性；E：KEGG通路分析Hsp90AA1在PI3K-Akt信号通路中的具体调控关系
Fig.3 PPI analysis of key protein interactions in Heat stroke
表2 核心基因差异表达结果
Tab.2 Differential expression results of core genes
基因 LogFC P值
Ddit3 -1.197289796 0.000980392
Sod1 -1.399359178 0.000361197
Jun -2.074465821 0.001547988
Hmox1 -2.092347052 0.001547988
Egr1 -2.096875439 0.001547988
Hsp90AA1 -2.270013815 0.001547988
Hsp90ab1 -2.369111015 0.000619195
Hspb1 -2.766033021 0.000980392
图4 Western blot检测Hsp90AA1在热射病患者中的表达A：生存组与死亡组Hsp 90AA1的表达；B：发病初期与发病后24 h的Hsp90AA1的表达
Fig.4 Expression of Hsp90AA1 in patients with febrile radiation
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