基于转录组学测序与生物信息学筛选低氧诱导小鼠肾脏炎症反应的关键基因并验证  

Screening and validation of key genes for hypoxia-induced renal inflammatory reaction in mice by transcriptome sequencing and bioinformatics

作　　者：许鑫桐 龙启福 胡英 贾茹涵 马如雪 永胜 XU Xintong;LONG Qifu;HU Ying;JIA Ruhan;MA Ruxue;YONG Sheng(Department of Basic Medicine,College of Medicine,Qinghai University,Xining 810016,China)

机构地区：[1]青海大学医学院基础医学部,西宁810016

出　　处：《中国医科大学学报》2024年第12期1071-1079,共9页Journal of China Medical University

基　　金：国家自然科学基金(82060295)。

摘　　要：目的利用转录组学测序与生物信息学挖掘低氧诱导小鼠肾脏炎症反应的关键基因并验证。方法于海拔4200 m和海拔400 m分别饲养C57/BL6小鼠,构建高原低氧组和平原常氧组小鼠模型,30 d后无菌取出小鼠肾脏组织,HE染色分析肾脏病理学变化,并测定低氧暴露下小鼠血气分析和肾脏指数改变。然后对高原低氧组和平原常氧组小鼠肾脏组织进行转录组学测序分析,并利用生物信息学技术筛选关键基因,通过实时定量逆转录PCR(RT-qPCR)和Western blotting对关键基因进行验证。结果HE染色结果显示与平原常氧组比较,高原低氧组小鼠肾小球萎缩,低氧暴露下小鼠血气分析和肾脏指数均下降。转录组学测序分析结果显示,高原低氧组中共有3007个差异表达基因,其中123个是与炎症相关的差异表达基因(IR-DEGs)。对IR-DEGs进行GO和KEGG富集分析结果显示,在细胞因子-细胞因子受体相互作用、趋化因子等炎症相关信号通路显著富集。对IR-DEGs构建蛋白质-蛋白质相互作用(PPI)网络结果显示,共鉴定出STAT3、TLR7、CD68、NFKBIA、LEP、APOE 6个Hub基因。RT-qPCR检测结果显示,6个Hub基因的mRNA表达均上调,与转录组测序结果一致。Western blotting检测结果显示,CD68、NFKBIA、LEP、TLR7和APOE蛋白表达上调,STAT3表达下调。结论STAT3、CD68、NFKBIA、LEP、TLR7和APOE是低氧诱导炎症反应的关键基因。低氧环境通过上调机体TLR7、CD68、STAT3、LEP、APOE的表达,诱导小鼠肾脏组织发生炎症反应。Objective To screen and validate the key genes involved in hypoxia-induced inflammatory reactions in mice using transcriptome sequencing and bioinformatics.Methods C57/BL6 mice were bred at altitudes of 4200 m and 400 m,and mouse models were constructed for the plateau hypoxia(HKT)group and the plain normoxia(PKC)group.Kidney tissues were aseptically removed after 30 days,renal pathological changes were analyzed by HE staining,blood gas analysis and renal index changes were measured in the mice under hypoxia.The kidney tissues of mice in the HKT and PKC groups were analyzed using transcriptome sequencing,key genes were screened using bioinformatics technology,and these genes were verified using real-time quantitative reverse transcription PCR(RT-qPCR)and Western blotting.Results HE staining showed glomerular atrophy in mice in the HKT group compared with the PKC group,and a decrease in blood gas analysis and renal index occurred in mice exposed to hypoxia.Transcriptome sequencing analysis revealed 3007 differentially expressed genes(DEGs)in the HKT group,of which 123 were inflammation-related DEGs(IR-DEGs).GO and KEGG enrichment analyses of IR-DEGs showed significant enrichment in inflammation-related signaling pathways,such as cytokine-cytokine receptor interactions and chemokines.The results of the protein-protein interaction(PPI)network construction of IR-DEGs showed that six hub genes,STAT3,TLR7,CD68,NFKBIA,LEP,and APOE,were identified,and the mRNA expression of these six genes was upregulated according to RT-qPCR results,which was in agreement with the results of transcriptome sequencing.Western blotting showed that CD68,NFKBIA,LEP,TLR7,and APOE expression was upregulated while STAT3 expression was downregulated.Conclusion STAT3,CD68,NFKBIA,LEP,TLR7,and APOE are the key genes involved in hypoxia-induced inflammatory reactions.A hypoxic environment induced inflammatory reactions in mouse kidney tissues by upregulating the expression of TLR7,CD68,STAT3,LEP,and APOE.

关 键 词：高原低氧 肾脏 炎症反应 转录组学测序 生物信息学 

分 类 号：R34[医药卫生—基础医学]