生物信息学鉴定低氧诱导小鼠肾脏线粒体损伤的Hub基因及其作用机制

doi:10.6040/j.issn.1671-7554.0.2023.0427

摘要/Abstract

摘要： 目的利用生物信息学分析挖掘低氧介导线粒体损伤的关键基因,并分析关键基因与小鼠肾脏组织凋亡的相关性,进一步阐明高原低氧条件下小鼠肾脏组织损伤的分子作用机制。方法构建30 d常氧与低氧动物模型,分别作为平原常氧组(n=5)和高原低氧组(n=5),测定血气分析指标与小鼠肾脏指数,通过H&E染色观察小鼠肾脏组织病理学改变,对平原常氧组与高原低氧组小鼠肾脏组织进行转录组学测序。随后将测序得到的差异表达基因(DEGs)分别与线粒体数据库、凋亡相关基因数据库进行关联,鉴定差异线粒体功能相关基因(DE-MFRGs)与差异凋亡相关基因(DE-ARGs),蛋白质互作(PPI)网络筛选Hub基因,利用生物信息学对DEGs、DE-MFRGs、DE-ARGs进行富集分析,并通过RT-qPCR 和 Western blotting对Hub 基因及凋亡基因进行验证。结果与平原常氧组相比,高原低氧组小鼠肾脏指数显著下降。H&E染色结果显示,高原低氧组小鼠肾小球明显萎缩,肾小管上皮细胞肿胀、破裂,炎细胞浸润,部分肾小管上皮细胞体积增大,胞浆疏松淡染,可见空泡变性。低氧暴露条件下共鉴定出3 007个DEGs,其中有464个基因与线粒体功能相关,被定义为DE-MFRGs。对DE-MFRGs进行KEGG富集分析发现,其主要富集在帕金森病信号通路、氧化磷酸化信号通路、产热信号通路、阿尔兹海默病信号通路、三羧酸循环信号通路、NAFLD信号通路、代谢信号通路等与线粒体功能相关的通路中。通过构建PPI网络共鉴定出3个Hub基因(SOD2、TUFM、MRPL12),SOD2、TUFM、MRPL12的mRNA和蛋白表达量均下调。进一步发现Hub基因与33个ARGs基因均出现正相关与负相关,并验证了小鼠肾脏组织ARGs中CASPASE3、BCL2、BAK基因的mRNA与蛋白表达量,结果显示,CASPASE3与BAK基因表达上调,BCL2基因表达下调。结论高原低氧环境通过调控SOD2、TUFM与MRPL12基因的表达,介导小鼠肾脏组织线粒体功能紊乱,进一步诱导小鼠肾脏组织损伤与凋亡。

关键词: 高原低氧, 肾脏, 线粒体功能, Hub基因, 凋亡, 线粒体数据库

Abstract: Objective To explore the Hub genes of hypoxia-induced mitochondrial damage by bioinformatics analysis, and to analyze the correlation between hub genes and apoptosis of mouse kidney tissue, so as to elucidate the molecular mechanism of hypoxia-induced mitochondrial damage in mice at high altitude. Methods After animal models of 30 d normoxicity group(n=5)and hypoxia group(n=5)were established, blood gas and renal indexes were determined. Histopathological changes of renal tissues were observed with H&E staining, and transcriptomics sequencing was performed. The differentially expressed genes(DEGs)were correlated with mitochondrial database and apoptosis-related gene database to identify DEGs related to mitochondrial function(DE-MFRGs)and DEGs related to apoptosis(DE-ARGs), and to screen Hub genes for protein-protein interaction(PPI). KEGG enrichment analysis was conducted on DEGs, DE-MFRGs and DE-ARGs. Hub genes and DE-ARGs were verified with RT-qPCR and Western blotting. Results Compared with the normoxicity group, the hypoxia group had significantly decreased kidney indexes. H&E staining results showed that the glomeruli in the hypoxia group were significantly atrophied; the renal tubule epithelial cells were swollen, ruptured, or infiltrated by inflammatory cells; the volume of some renal tubule epithelial cells was increased; the cytoplasm was loose and light stained, with cavitation degeneration. A total of 3,007 DEGs were identified under hypoxic exposure, among which 464 were related to mitochondrial function and were defined as DE-MFRGs. KEGG enrichment analysis of DE-MFRGs showed that they were involved with Parkinsons disease signaling pathway, oxidative phosphorylation signaling pathway, thermogenesis signaling pathway, Alzheimers disease signaling pathway, tricarboxylic acid cycling signaling pathway, NAFLD signaling pathway, metabolic signaling pathway and other pathways related to mitochondrial function. Three Hub genes(SOD2, TUFM and MRPL12)were identified through the construction of PPI network. The mRNA and protein expressions of SOD2, TUFM and MRPL12 were down-regulated. Furthermore, Hub genes were positively or negatively correlated with 33 ARGs genes, and the mRNA expression of CASPASE3 and BAK were up-regulated, while that of BCL2 was down-regulated. Conclusion The high altitude hypoxia environment can regulate the mRNA expressions of SOD2, TUFM and MRPL12, mediate the mitochondrial dysfunction of mouse kidney tissue, and induce damage and apoptosis.

Key words: Plateau hypoxia, Kidney, Mitochondrial function, Hub gene, Apoptosis, Mitochondrial database

中图分类号:

高玉杰,龙启福,胡英,许玉珍,王茹,永胜. 生物信息学鉴定低氧诱导小鼠肾脏线粒体损伤的Hub基因及其作用机制[J]. 山东大学学报 (医学版), 2023, 61(9): 57-68.

GAO Yujie, LONG Qifu, HU Ying, XU Yuzhen, WANG Ru, YONG Sheng. Bioinformatics identification of the Hub genes and mechanism of hypoxia-induced mitochondrial damage in mouse kidney[J]. Journal of Shandong University (Health Sciences), 2023, 61(9): 57-68.

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