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

doi:10.12307/2023.734

Abstract

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

CLC Number:

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

Liang Xuezhen, Luo Di, Li Jiacheng, Wen Mingtao, Zhang Jian, Xu Bo, Li Gang. PTGS2 and STAT3 in steroid-induced osteonecrosis of the femoral head: ferroptosis-related potential diagnostic biomarkers[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(36): 5898-5904.

Figures/Tables 9

References

[1] HAUZEUR JP, MALAISE M, DE MAERTELAER V. A prospective cohort study of the clinical presentation of non-traumatic osteonecrosis of the femoral head: spine and knee symptoms as clinical presentation of hip osteonecrosis. Int Orthop. 2016;40(7):1347-1351.
[2] ZHANG QY, LI ZR, GAO FQ, et al. Pericollapse Stage of Osteonecrosis of the Femoral Head: A Last Chance for Joint Preservation. Chin Med J (Engl). 2018; 131(21):2589-2598.
[3] MONT MA, ZYWIEL MG, MARKER DR, et al. The natural history of untreated asymptomatic osteonecrosis of the femoral head: a systematic literature review. J Bone Joint Surg Am. 2010;92(12):2165-2170.
[4] 傅维民,刘保一,王本杰,等.激素性股骨头坏死的保髋治疗策略及疗效[J].中华骨科杂志,2019,39(23):1424-1431.
[5] LIANG XZ, LUO D, CHEN YR, et al. Identification of potential autophagy-related genes in steroid-induced osteonecrosis of the femoral head via bioinformatics analysis and experimental verification. J Orthop Surg Res. 2022;17(1):86.
[6] COOPER C, STEINBUCH M, STEVENSON R, et al. The epidemiology of osteonecrosis: findings from the GPRD and THIN databases in the UK. Osteoporos Int. 2010;21(4):569-577.
[7] HADIAN K, STOCKWELL BR. SnapShot: Ferroptosis. Cell. 2020;181(5):1188-1188.e1.
[8] CHEN K, LIU Y, HE J, et al. Steroid-induced osteonecrosis of the femoral head reveals enhanced reactive oxygen species and hyperactive osteoclasts. Int J Biol Sci. 2020;16(11):1888-1900.
[9] LU J, YANG J, ZHENG Y, et al. Extracellular vesicles from endothelial progenitor cells prevent steroid-induced osteoporosis by suppressing the ferroptotic pathway in mouse osteoblasts based on bioinformatics evidence. Sci Rep. 2019; 9(1):16130.
[10] 章喻,王利波,戴薇薇.糖皮质激素与铁死亡[J].生理科学进展,2022,53(1): 76-81.
[11] LI T, ZHANG Y, WANG R, et al. Discovery and validation an eight-biomarker serum gene signature for the diagnosis of steroid-induced osteonecrosis of the femoral head. Bone. 2019;122:199-208.
[12] ZHOU N, BAO J. FerrDb: a manually curated resource for regulators and markers of ferroptosis and ferroptosis-disease associations. Database (Oxford). 2020;2020:baaa021.
[13] ASHBURNER M, BALL CA, BLAKE JA, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25(1):25-29.
[14] KANEHISA M, GOTO S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27-30.
[15] SZKLARCZYK D, GABLE AL, NASTOU KC, et al.The STRING database in 2021: customizable protein–protein networks, and functional characterization of user-uploaded gene/measurement sets. Nucleic Acids Res. 2021;49(D1):D605-D612
[16] BADER GD, HOGUE CW. An automated method for finding molecular complexes in large protein interaction networks. BMC Bioinformatics. 2003;4:2.
[17] XUE J, CHEN L, CHENG H, et al. The Identification and Validation of Hub Genes Associated with Acute Myocardial Infarction Using Weighted Gene Co-Expression Network Analysis. J Cardiovasc Dev Dis. J 2022;9(1):30.
[18] ZHAO D W, YU M, HU K, et al. Prevalence of Nontraumatic Osteonecrosis of the Femoral Head and its Associated Risk Factors in the Chinese Population: Results from a Nationally Representative Survey. Chin Med J (Engl). 2015;128(21):2843-2850.
[19] MURAKAMI J, ISHII M, SUEHIRO F, et al. Vascular endothelial growth factor-C induces osteogenic differentiation of human mesenchymal stem cells through the ERK and RUNX2 pathway. Biochem Biophys Res Commun. 2017;484(3):710-718.
[20] RUDMAN HA, BIRRELL F, PEARCE MS, et al. Obesity, bone density relative to body weight and prevalent vertebral fracture at age 62 years: the Newcastle thousand families study. Osteoporos Int. 2019;30(4):829-836.
[21] 陈彦同, 周明旺, 李盛华, 等.基于“髓病理论”的非创伤性股骨头坏死发病机理探析[J].时珍国医国药,2020,31(7):1684-1686.
[22] HUANG Z, WANG Q, ZHANG T, et al. Hyper-activated platelet lysates prevent glucocorticoid-associated femoral head necrosis by regulating autophagy. Biomed Pharmacother. 2021;139:111711.
[23] PETEK D, HANNOUCHE D, SUVA D. Osteonecrosis of the femoral head: pathophysiology and current concepts of treatment. EFORT Open Rev. 2019;4(3): 85-97.
[24] LIU GB, LI R, LU Q, et al. Three-dimensional distribution of cystic lesions in osteonecrosis of the femoral head. J Orthop Translat. 2020;22:109-115.
[25] YOUM YS, LEE SY, LEE SH. Apoptosis in the osteonecrosis of the femoral head. Clin Orthop Surg. 2010;2(4):250-255.
[26] DIXON SJ, LEMBERG KM, LAMPRECHT MR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060-1072.
[27] ZHENG J, CONRAD M. The Metabolic Underpinnings of Ferroptosis. Cell Metab. 2020;32(6):920-937.
[28] ZHENG H, JIANG J, XU S, et al. Nanoparticle-induced ferroptosis: detection methods, mechanisms and applications. Nanoscale. 2021;13(4):2266-2285.
[29] OKAZAKI S, NAGOYA S, MATSUMOTO H, et al. TLR4 stimulation and corticosteroid interactively induce osteonecrosis of the femoral head in rat. J Orthop Res. 2016; 34(2):342-345.
[30] ZHU D, YU H, LIU P, et al. Calycosin modulates inflammation via suppressing TLR4/NF-κB pathway and promotes bone formation to ameliorate glucocorticoid-induced osteonecrosis of the femoral head in rat. Phytother Res. 2021. doi: 10.1002/ptr.7028.
[31] KUBO Y, DRESCHER W, FRAGOULIS A, et al. Adverse Effects of Oxidative Stress on Bone and Vasculature in Corticosteroid-Associated Osteonecrosis: Potential Role of Nuclear Factor Erythroid 2-Related Factor 2 in Cytoprotection. Antioxid Redox Signal. 2021;35(5):357-376.
[32] FAN ZQ, BAI SC, XU Q, et al. Oxidative Stress Induced Osteocyte Apoptosis in Steroid-Induced Femoral Head Necrosis. Orthop Surg. 2021;13(7):2145-2152.
[33] SHI W, ZHANG X, XU C, et al. Identification of Hub Genes and Pathways Associated with Oxidative Stress of Cartilage in Osteonecrosis of Femoral Head Using Bioinformatics Analysis. Cartilage. 2022;13(1):19476035221074000.
[34] LIU S, DU J, LI D, et al. Oxidative stress induced pyroptosis leads to osteogenic dysfunction of MG63 cells. J Mol Histol. 2020;51(3):221-232.
[35] WU Y, WANG J, ZHAO T, et al. Di-(2-ethylhexyl) phthalate exposure leads to ferroptosis via the HIF-1α/HO-1 signaling pathway in mouse testes. J Hazard Mater. 2022;426:127807.
[36] STAINES BOONE AT, ALCÁNTARA-MONTIEL JC, SÁNCHEZ-SÁNCHEZ LM, et al. Zoledronate as effective treatment for minimal trauma fractures in a child with STAT3 deficiency and osteonecrosis of the hip. Pediatr Blood Cancer. 2016; 63(11):2054-2057.
[37] WU Y, FANG L, GAO Y, et al. lncRNA FGD5-AS1 Regulates Bone Marrow Stem Cell Proliferation and Apoptosis by Affecting miR-296-5p/STAT3 Axis in Steroid-Induced Osteonecrosis of the Femoral Head. J Healthc Eng. 2022;2022:9364467.
[38] JIANG HT, RAN CC, LIAO YP, et al. IGF-1 reverses the osteogenic inhibitory effect of dexamethasone on BMP9-induced osteogenic differentiation in mouse embryonic ﬁbroblasts via PI3K/AKT/COX-2 pathway. J Steroid Biochem Mol Biol. 2019;191:105363.