m6A-related gene clustering analysis and immune cell infiltration analysis in myocardial ischemia-reperfusion injury after cardiopulmonary bypass based on machine learning_Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Authors：

TANG Yao , CHEN Wendong , WANG Yanqiong ,  YANG Wei

Department of Anesthesiology, The First Affiliated Hospital of Kunming Medical University, Kunming, 650032, P. R. China;

Corresponding author：

YANG Wei, Email: ywkm0@163.com

Keywords：

Cardiopulmonary bypass; N6-methyladenosine (m6A) RNA methylation; myocardial ischemia-reperfusion injury; machine learning

DOI：

10.7507/1007-4848.202312072

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Objective To identify the N6-methyladenosine (m6A)-related characteristic genes analyzed by gene clustering and immune cell infiltration in myocardial ischemia-reperfusion injury (MI/RI) after cardiopulmonary bypass through machine learning. Methods The differential genes associated with m6A methylation were screened by the dataset GSE132176 in GEO, the samples of the dataset were clustered based on the differential gene expression profile, and the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differential genes of the m6A cluster after clustering were performed to determine the gene function of the m6A cluster. R software was used to determine the better models in machine learning of support vector machine (SVM) model and random forest (RF) model, which were used to screen m6A-related characteristic genes in MI/RI, and construct characteristic gene nomogram to predict the incidence of disease. R software was used to analyze the correlation between characteristic genes and immune cells, and the online website was used to build a characteristic gene regulatory network. Results In this dataset, a total of 5 m6A-related differential genes were screened, and the gene expression profiles were divided into two clusters for cluster analysis. The enrichment analysis of m6A clusters showed that these genes were mainly involved in regulating monocytes differentiation, response to lipopolysaccharides, response to bacteria-derived molecules, cellular response to decreased oxygen levels, DNA transcription factor binding, DNA-binding transcription activator activity, RNA polymerase Ⅱ specificity, NOD-like receptor signaling pathway, fluid shear stress and atherosclerosis, tumor necrosis factor signaling pathway, interleukin-17 signaling pathway. The RF model was determined by R software as the better model, which determined that METTL3, YTHDF1, RBM15B and METTL14 were characteristic genes of MI/RI, and mast cells, type 1 helper lymphocytes (Th1), type 17 helper lymphocytes (Th17), and macrophages were found to be associated with MI/RI after cardiopulmonary bypass in immune cell infiltration. Conclusion The four characteristic genes METTL3, YTHDF1, RBM15B and METTL14 are obtained by machine learning, while cluster analysis and immune cell infiltration analysis can better reveal the pathophysiological process of MI/RI.

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2.	Liu Y, Yang D, Liu T, et al. N6-methyladenosine-mediated gene regulation and therapeutic implications. Trends Mol Med, 2023, 29(6): 454-467.
3.	Fu Y, Dominissini D, Rechavi G, et al. Gene expression regulation mediated through reversible m⁶A RNA methylation. Nat Rev Genet, 2014, 15(5): 293-306.
4.	Yang Y, Hsu PJ, Chen YS, et al. Dynamic transcriptomic m6A decoration: Writers, erasers, readers and functions in RNA metabolism. Cell Res, 2018, 28(6): 616-624.
5.	Li J, Zhang H, Wang H. N1-methyladenosine modification in cancer biology: Current status and future perspectives. Comput Struct Biotechnol J, 2022, 20: 6578-6585.
6.	Xu Z, Lv B, Qin Y, et al. Emerging roles and mechanism of m6a methylation in cardiometabolic diseases. Cells, 2022, 11(7): 1101.
7.	Mathiyalagan P, Adamiak M, Mayourian J, et al. FTO-dependent N6-methyladenosine regulates cardiac function during remodeling and repair. Circulation, 2019, 139(4): 518-532.
8.	Dorn LE, Lasman L, Chen J, et al. The N6-methyladenosine mRNA methylase METTL3 controls cardiac homeostasis and hypertrophy. Circulation, 2019, 139(4): 533-545.
9.	Berulava T, Buchholz E, Elerdashvili V, et al. Changes in m6A RNA methylation contribute to heart failure progression by modulating translation. Eur J Heart Fail, 2020, 22(1): 54-66.
10.	Jiang X, Liu B, Nie Z, et al. The role of m6A modification in the biological functions and diseases. Signal Transduct Target Ther, 2021, 6(1): 74.
11.	Song N, Cui K, Zhang K, et al. The role of m6A RNA methylation in cancer: Implication for nature products anti-cancer research. Front Pharmacol, 2022, 13: 933332.
12.	魏成露, 冯庆敏, 陈宋程, 等. 心肌缺血再灌注损伤分子机制的研究进展. 海南医学院学报, 2022, 28(16): 1268-1274.Wei CL, Feng QM, Chen SC, et al. Advances in molecular mechanism of myocardial ischemia/reperfusion injury. J Hainan Med Univ, 2022, 28(16): 1268-1274.
13.	Chen DH, Zhang JG, Wu CX, et al. Non-coding RNA m6A modification in cancer: Mechanisms and therapeutic targets. Front Cell Dev Biol, 2021, 9: 778582.
14.	van der Laan AM, Ter Horst EN, Delewi R, et al. Monocyte subset accumulation in the human heart following acute myocardial infarction and the role of the spleen as monocyte reservoir. Eur Heart J, 2014, 35(6): 376-385.
15.	He Z, Ma C, Yu T, et al. Activation mechanisms and multifaceted effects of mast cells in ischemia reperfusion injury. Exp Cell Res, 2019, 376(2): 227-235.
16.	Bhattacharya K, Farwell K, Huang M, et al. Mast cell deficient W/Wv mice have lower serum IL-6 and less cardiac tissue necrosis than their normal littermates following myocardial ischemia-reperfusion. Int J Immunopathol Pharmacol, 2007, 20(1): 69-74.
17.	Frangogiannis NG, Lindsey ML, Michael LH, et al. Resident cardiac mast cells degranulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation, 1998, 98(7): 699-710.
18.	Chen S, Mu D, Cui M, et al. Dynamic changes and clinical significance of serum tryptase levels in STEMI patients treated with primary PCI. Biomarkers, 2014, 19(7): 620-624.
19.	Kwon JS, Kim YS, Cho AS, et al. The novel role of mast cells in the microenvironment of acute myocardial infarction. J Mol Cell Cardiol, 2011, 50(5): 814-825.
20.	Dohi T, Fujihashi K. Type 1 and 2 T helper cell-mediated colitis. Curr Opin Gastroenterol, 2006, 22(6): 651-657.
21.	Romagnani S. T-cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol, 2000, 85(1): 9-18.
22.	Li D, Yang Z, Gao S, et al. Tanshinone ⅡA ameliorates myocardial ischemia/reperfusion injury in rats by regulation of NLRP3 inflammasome activation and Th17 cells differentiation. Acta Cir Bras, 2022, 37(7): e370701.
23.	Chang SL, Hsiao YW, Tsai YN, et al. Interleukin-17 enhances cardiac ventricular remodeling via activating MAPK pathway in ischemic heart failure. J Mol Cell Cardiol, 2018, 122: 69-79.
24.	Zhang Z, Tang J, Cui X, et al. New insights and novel therapeutic potentials for macrophages in myocardial infarction. Inflammation, 2021, 44(5): 1696-1712.
25.	Frangogiannis NG. The inflammatory response in myocardial injury, repair, and remodelling. Nat Rev Cardiol, 2014, 11(5): 255-265.
26.	Kanisicak O, Khalil H, Ivey MJ, et al. Genetic lineage tracing defines myofibroblast origin and function in the injured heart. Nat Commun, 2016, 7: 12260.
27.	Wang X, Li Y, Li J, et al. Mechanism of METTL3-mediated m6A modification in cardiomyocyte pyroptosis and myocardial ischemia-reperfusion injury. Cardiovasc Drugs Ther, 2023, 37(3): 435-448.
28.	Tang J, Tang QX, Liu S. METTL3-modified lncRNA-SNHG8 binds to PTBP1 to regulate ALAS2 expression to increase oxidative stress and promote myocardial infarction. Mol Cell Biochem, 2023, 478(6): 1217-1229.
29.	Wu C, Chen Y, Wang Y, et al. The m6A methylation enzyme METTL14 regulates myocardial ischemia/reperfusion injury through the Akt/mTOR signaling pathway. Mol Cell Biochem, 2024, 479(6): 1391-1400.
30.	Wang H, Fu L, Li Y, et al. m6A methyltransferase WTAP regulates myocardial ischemia reperfusion injury through YTHDF1/FOXO3a signaling. Apoptosis, 2023, 28(5-6): 830-839.
31.	Loyer P, Busson A, Trembley JH, et al. The RNA binding motif protein 15B (RBM15B/OTT3) is a functional competitor of serine-arginine (SR) proteins and antagonizes the positive effect of the CDK11p110-cyclin L2α complex on splicing. J Biol Chem, 2011, 286(1): 147-159.
32.	Hiriart E, Gruffat H, Buisson M, et al. Interaction of the Epstein-Barr virus mRNA export factor EB2 with human Spen proteins SHARP, OTT1, and a novel member of the family, OTT3, links Spen proteins with splicing regulation and mRNA export. J Biol Chem, 2005, 280(44): 36935-36945.

1. Passaroni AC, Silva MA, Yoshida WB. Cardiopulmonary bypass: Development of John Gibbon's heart-lung machine. Rev Bras Cir Cardiovasc, 2015, 30(2): 235-245.
2. Liu Y, Yang D, Liu T, et al. N6-methyladenosine-mediated gene regulation and therapeutic implications. Trends Mol Med, 2023, 29(6): 454-467.
3. Fu Y, Dominissini D, Rechavi G, et al. Gene expression regulation mediated through reversible m⁶A RNA methylation. Nat Rev Genet, 2014, 15(5): 293-306.
4. Yang Y, Hsu PJ, Chen YS, et al. Dynamic transcriptomic m6A decoration: Writers, erasers, readers and functions in RNA metabolism. Cell Res, 2018, 28(6): 616-624.
5. Li J, Zhang H, Wang H. N1-methyladenosine modification in cancer biology: Current status and future perspectives. Comput Struct Biotechnol J, 2022, 20: 6578-6585.
6. Xu Z, Lv B, Qin Y, et al. Emerging roles and mechanism of m6a methylation in cardiometabolic diseases. Cells, 2022, 11(7): 1101.
7. Mathiyalagan P, Adamiak M, Mayourian J, et al. FTO-dependent N6-methyladenosine regulates cardiac function during remodeling and repair. Circulation, 2019, 139(4): 518-532.
8. Dorn LE, Lasman L, Chen J, et al. The N6-methyladenosine mRNA methylase METTL3 controls cardiac homeostasis and hypertrophy. Circulation, 2019, 139(4): 533-545.
9. Berulava T, Buchholz E, Elerdashvili V, et al. Changes in m6A RNA methylation contribute to heart failure progression by modulating translation. Eur J Heart Fail, 2020, 22(1): 54-66.
10. Jiang X, Liu B, Nie Z, et al. The role of m6A modification in the biological functions and diseases. Signal Transduct Target Ther, 2021, 6(1): 74.
11. Song N, Cui K, Zhang K, et al. The role of m6A RNA methylation in cancer: Implication for nature products anti-cancer research. Front Pharmacol, 2022, 13: 933332.
12. 魏成露, 冯庆敏, 陈宋程, 等. 心肌缺血再灌注损伤分子机制的研究进展. 海南医学院学报, 2022, 28(16): 1268-1274.Wei CL, Feng QM, Chen SC, et al. Advances in molecular mechanism of myocardial ischemia/reperfusion injury. J Hainan Med Univ, 2022, 28(16): 1268-1274.
13. Chen DH, Zhang JG, Wu CX, et al. Non-coding RNA m6A modification in cancer: Mechanisms and therapeutic targets. Front Cell Dev Biol, 2021, 9: 778582.
14. van der Laan AM, Ter Horst EN, Delewi R, et al. Monocyte subset accumulation in the human heart following acute myocardial infarction and the role of the spleen as monocyte reservoir. Eur Heart J, 2014, 35(6): 376-385.
15. He Z, Ma C, Yu T, et al. Activation mechanisms and multifaceted effects of mast cells in ischemia reperfusion injury. Exp Cell Res, 2019, 376(2): 227-235.
16. Bhattacharya K, Farwell K, Huang M, et al. Mast cell deficient W/Wv mice have lower serum IL-6 and less cardiac tissue necrosis than their normal littermates following myocardial ischemia-reperfusion. Int J Immunopathol Pharmacol, 2007, 20(1): 69-74.
17. Frangogiannis NG, Lindsey ML, Michael LH, et al. Resident cardiac mast cells degranulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation, 1998, 98(7): 699-710.
18. Chen S, Mu D, Cui M, et al. Dynamic changes and clinical significance of serum tryptase levels in STEMI patients treated with primary PCI. Biomarkers, 2014, 19(7): 620-624.
19. Kwon JS, Kim YS, Cho AS, et al. The novel role of mast cells in the microenvironment of acute myocardial infarction. J Mol Cell Cardiol, 2011, 50(5): 814-825.
20. Dohi T, Fujihashi K. Type 1 and 2 T helper cell-mediated colitis. Curr Opin Gastroenterol, 2006, 22(6): 651-657.
21. Romagnani S. T-cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol, 2000, 85(1): 9-18.
22. Li D, Yang Z, Gao S, et al. Tanshinone ⅡA ameliorates myocardial ischemia/reperfusion injury in rats by regulation of NLRP3 inflammasome activation and Th17 cells differentiation. Acta Cir Bras, 2022, 37(7): e370701.
23. Chang SL, Hsiao YW, Tsai YN, et al. Interleukin-17 enhances cardiac ventricular remodeling via activating MAPK pathway in ischemic heart failure. J Mol Cell Cardiol, 2018, 122: 69-79.
24. Zhang Z, Tang J, Cui X, et al. New insights and novel therapeutic potentials for macrophages in myocardial infarction. Inflammation, 2021, 44(5): 1696-1712.
25. Frangogiannis NG. The inflammatory response in myocardial injury, repair, and remodelling. Nat Rev Cardiol, 2014, 11(5): 255-265.
26. Kanisicak O, Khalil H, Ivey MJ, et al. Genetic lineage tracing defines myofibroblast origin and function in the injured heart. Nat Commun, 2016, 7: 12260.
27. Wang X, Li Y, Li J, et al. Mechanism of METTL3-mediated m6A modification in cardiomyocyte pyroptosis and myocardial ischemia-reperfusion injury. Cardiovasc Drugs Ther, 2023, 37(3): 435-448.
28. Tang J, Tang QX, Liu S. METTL3-modified lncRNA-SNHG8 binds to PTBP1 to regulate ALAS2 expression to increase oxidative stress and promote myocardial infarction. Mol Cell Biochem, 2023, 478(6): 1217-1229.
29. Wu C, Chen Y, Wang Y, et al. The m6A methylation enzyme METTL14 regulates myocardial ischemia/reperfusion injury through the Akt/mTOR signaling pathway. Mol Cell Biochem, 2024, 479(6): 1391-1400.
30. Wang H, Fu L, Li Y, et al. m6A methyltransferase WTAP regulates myocardial ischemia reperfusion injury through YTHDF1/FOXO3a signaling. Apoptosis, 2023, 28(5-6): 830-839.
31. Loyer P, Busson A, Trembley JH, et al. The RNA binding motif protein 15B (RBM15B/OTT3) is a functional competitor of serine-arginine (SR) proteins and antagonizes the positive effect of the CDK11p110-cyclin L2α complex on splicing. J Biol Chem, 2011, 286(1): 147-159.
32. Hiriart E, Gruffat H, Buisson M, et al. Interaction of the Epstein-Barr virus mRNA export factor EB2 with human Spen proteins SHARP, OTT1, and a novel member of the family, OTT3, links Spen proteins with splicing regulation and mRNA export. J Biol Chem, 2005, 280(44): 36935-36945.

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