中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (36): 5898-5904.doi: 10.12307/2023.734

• 骨与关节综述 bone and joint review • 上一篇    

激素性股骨头坏死中的PTGS2和STAT3:潜在铁死亡相关诊断生物标志物

梁学振1,2,骆  帝2,李嘉程1,温明韬1,张  建1,许  波1,2,李  刚1,2   

  1. 1山东中医药大学第一临床医学院,山东省济南市   250355;2山东中医药大学附属医院显微骨科,山东省济南市   250014
  • 收稿日期:2022-10-08 接受日期:2022-11-25 出版日期:2023-12-28 发布日期:2023-03-27
  • 通讯作者: 李刚,博士,教授,主任医师,山东中医药大学第一临床医学院,山东省济南市 250355;山东中医药大学附属医院显微骨科,山东省济南市 250014 许波,博士,副教授,副主任医师,山东中医药大学第一临床医学院,山东省济南市 250355;山东中医药大学附属医院显微骨科,山东省济南市 250014
  • 作者简介:梁学振,男,1990年生,山东省新泰市人,汉族,2019年山东中医药大学毕业,博士,副教授,主要从事中医药防治骨坏死、代谢性骨病的临床与基础研究。
  • 基金资助:
    国家自然科学基金资助项目(82205154),项目负责人:梁学振;山东省自然科学基金青年项目(ZR2021QH004),项目负责人:梁学振;济南市临床医学科技创新计划(202019056),项目负责人:梁学振;山东省中医药科技项目(2020Q009),项目负责人:梁学振

PTGS2 and STAT3 in steroid-induced osteonecrosis of the femoral head: ferroptosis-related potential diagnostic biomarkers

Liang Xuezhen1, 2, Luo Di2, Li Jiacheng1, Wen Mingtao1, Zhang Jian1, Xu Bo1, 2, Li Gang1, 2   

  1. 1First Clinical Medical School, Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong Province, China; 2Department of Orthopedic Microsurgery, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China
  • Received:2022-10-08 Accepted:2022-11-25 Online:2023-12-28 Published:2023-03-27
  • Contact: Li Gang, MD, Professor, Chief physician, First Clinical Medical School, Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong Province, China; Department of Orthopedic Microsurgery, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China Xu Bo, MD, Associate professor, Associate chief physician, First Clinical Medical School, Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong Province, China; Department of Orthopedic Microsurgery, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China
  • About author:Liang Xuezhen, MD, Associate professor, First Clinical Medical School, Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong Province, China; Department of Orthopedic Microsurgery, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 82205154 (to LXZ); Shandong Natural Science Foundation Youth Program, No. ZR2021QH004 (to LXZ); Jinan Clinical Medical Science and Technology Innovation Plan, No. 202019056 (to LXZ); Shandong Traditional Chinese Medicine Science and Technology Project, No. 2020Q009 (to LXZ)

摘要:


文题释义:

激素性股骨头坏死:激素应用已成为继创伤之后引起股骨头坏死的第二大原因,激素使股骨头内多种细胞死亡,大部分激素性骨坏死发生在用药后的2年内,主要集中在6个月内,后期甚至引起股骨头塌陷,已成为导致髋关节残疾最常见的原因之一。若早期诊断与防治有望取得良好的临床疗效,因此,寻找可靠的诊断生物标志物是目前亟待解决的问题。
铁死亡(Ferroptosis):是最早由哥伦比亚大学Dr. Brent R.Stockwell在2012年提出的,一种铁依赖性的、细胞内脂质活性氧累积的、区别于凋亡、焦亡、自噬的新型细胞程序性死亡方式;其本质是谷胱甘肽的耗竭,谷胱甘肽过氧化物酶(GPX4)活性下降,脂质氧化物不能通过GPX4催化的谷胱甘肽还原酶反应代谢,之后二价的铁离子氧化脂质产生活性氧,从而促使铁死亡的发生。

背景:研究推测铁死亡可能参与了激素性股骨头坏死的病理过程,但铁死亡在激素性股骨头坏死中的发病机制尚不清楚。
目的:旨在通过生物信息学手段分析激素性股骨头坏死进展过程中调控铁死亡相关的关键基因,以进一步阐明铁死亡在激素性股骨头坏死中的生物学机制。
方法:从基因表达综合数据库(GEO)下载血清来源的GSE123568数据集,包括10个非激素性股骨头坏死(类固醇给药后)样本和30个激素性股骨头坏死样本;从FerrDb数据库整理铁死亡相关基因。通过将GSE123568数据集与铁死亡基因集映射,筛选激素性股骨头坏死中铁死亡相关的差异基因,通过“clusterProfiler”R包分析差异基因的基因本体论(GO)和京都基因和基因组百科全书(KEGG)通路富集,通过“corrplot”R包分析差异基因的Spearman相关系数,通过STRING数据库构建蛋白质-蛋白质相互作用(PPI)网络,通过MCODE插件权重分析以识别重要的基因簇模块,通过CytoHubba插件利用拓扑网络算法筛选Hub基因,通过“pROC”R包分析Hub基因的ROC曲线;借助髋关节软骨来源的GSE74089数据集验证Hub基因的表达水平。

结果与结论:①根据调整后的P值<0.01和|log2 FC| >log21.5的既定标准,在非激素性股骨头坏死和激素性股骨头坏死患者的外周血样本中筛选出30个激素性股骨头坏死中铁死亡相关的差异基因,包括20个上调基因和10个下调基因;②GO和KEGG通路富集分析表明,差异基因主要富集在氧化应激、缺氧诱导因子1信号通路和铁死亡;Spearman相关性分析显示差异基因之间存在一定的相关性;③PPI网络揭示差异基因在生物系统中具有相互作用关系,通过MCODE插件识别出2个重要的基因簇模块,通过CytoHubba插件的MCC,MNC,DMNC,Degree和EPC算法交叉映射确定7个Hub基因;④ROC曲线表明与非激素性股骨头坏死样本相比,Hub基因在激素性股骨头坏死样本中具有更高的诊断价值;⑤GSE74089数据集交叉验证明确激素性股骨头坏死髋关节软骨标本中PTGS2和STAT3的表达水平相对于正常样本显著升高,这与GSE123568数据集血清样本表达一致;⑥结论:通过生物信息学分析挖掘出30个激素性股骨头坏死中铁死亡相关的差异基因,通过构建其PPI网络和外部数据集交叉验证,筛选得到PTGS2和STAT3有望成为潜在诊断生物标志物的Hub基因,为进一步深入探索激素性股骨头坏死铁死亡相关的作用机制和诊断提供靶点和新的见解。

https://orcid.org/0000-0001-5649-4212 (梁学振); 

https://orcid.org/0000-0002-4587-6650 (李刚); 

https://orcid.org/0000-0003-1863-4530 (许波) 

中国组织工程研究杂志出版内容重点:人工关节;骨植入物;脊柱;骨折;内固定;数字化骨科;组织工程

关键词: 激素性股骨头坏死, 铁死亡, 生物信息学, 氧化应激, 生物标志物

Abstract: BACKGROUND: Previous experimental studies have shown that ferroptosis might be involved in the pathological process of steroid-induced osteonecrosis of the femoral head, but the pathogenesis of ferroptosis in steroid-induced osteonecrosis of the femoral head remains unclear.  
OBJECTIVE: To analyze the key potential ferroptosis-related genes involved in steroid-induced osteonecrosis of the femoral head to further clarify the biological mechanism of ferroptosis in steroid-induced osteonecrosis of the femoral head by bioinformatics.
METHODS: The GSE123568 mRNA expression profile dataset, including 10 non-steroid-induced osteonecrosis of the femoral head (following steroid administration) samples and 30 steroid-induced osteonecrosis of the femoral head samples, was downloaded from the Gene Expression Omnibus (GEO) database. Ferroptosis-related genes were obtained from the Human Ferroptosis Database (FerrDb). The ferroptosis-related genes involved in steroid-induced osteonecrosis of the femoral head were screened by intersecting the GSE123568 dataset with the set of ferroptosis genes. The differentially expressed ferroptosis-related genes involved in steroid-induced osteonecrosis of the femoral head were identified with R software. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the differentially expressed ferroptosis-related genes involved in steroid-induced osteonecrosis of the femoral head were conducted by “clusterProfiler” R package. Spearman correlations between the expression levels of the differentially expressed ferroptosis-related genes involved in steroid-induced osteonecrosis of the femoral head were confirmed with “corrplot” R package. Moreover, the protein-protein interaction (PPI) network was analyzed by using the Search Tool for the Retrieval of Interacting Genes (STRING); significant gene cluster modules were identified with the MCODE Cytoscape plugin, and hub genes among the differentially expressed ferroptosis-related genes involved in steroid-induced osteonecrosis of the femoral head were screened by CytoHubba plugin. The ROC curves of the final specifically expressed hub genes were analyzed by “pROC” R package. Finally, the expression levels of the hub genes of the differentially expressed ferroptosis-related genes involved in steroid-induced osteonecrosis of the femoral head were validated by using the GSE74089 dataset.  
RESULTS AND CONCLUSION: (1) A total of 30 differentially expressed ferroptosis-related genes were identified between the peripheral blood samples of steroid-induced osteonecrosis of the femoral head patients and non-steroid-induced osteonecrosis of the femoral head patients based on the defined criteria of adjusted P value <0.01 and |log2 FC| >log21.5, including 20 upregulated genes and 10 downregulated genes. (2) The GO and KEGG pathway enrichment analyses revealed that these differentially expressed ferroptosis-related genes were particularly enriched in oxidative stress, hypoxia-inducible factor-1 signaling pathway and ferroptosis. Spearman correlation analysis revealed significant correlations among the differentially expressed ferroptosis-related genes. (3) The PPI results demonstrated that the differentially expressed ferroptosis-related genes interacted with each other. Two significant gene cluster modules were identified through the MCODE plugin, and seven hub genes were identified by using the intersecting results within the MCC, MNC, DMNC, Degree and EPC algorithms of CytoHubba. (4) ROC curve suggested that compared to non-steroid-induced osteonecrosis of the femoral head samples, these seven overlapped ferroptosis-related hub genes had higher diagnostic value in the steroid-induced osteonecrosis of the femoral head samples. (5) The GSE74089 dataset showed that PTGS2 and STAT3 were significantly upregulated in the hip cartilage specimens, which was consistent with the GSE123568 dataset. (6) It is concluded that thirty potential ferroptosis-related genes were identified via bioinformatics analysis. The PPI network was constructed to select the Hub genes in which PTGS2 and STAT3 might serve as potential diagnostic biomarkers because they regulated ferroptosis. These results provide a target and new insight for further exploring the ferroptosis-related action mechanism and diagnosis of steroid-induced osteonecrosis of the femoral head.

Key words: steroid-induced osteonecrosis of the femoral head, ferroptosis, bioinformatics, oxidative stress, biomarker

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