Inhibiting miR-155 protects against myocardial ischemia/reperfusion injury via targeted regulation of HIF-1α in rats

Document Type: Original Article

Authors

1 Department of Pediatric Surgery, Jingzhou Central Hospital, the Second Clinical Medical College, Yangtze University, Jingzhou 434020, Hubei, China

2 Department of Orthopedic surgery, Jingzhou Central Hospital, the Second Clinical Medical College, Yangtze University, Jingzhou 434020, Hubei, China

3 Department of Clinical Laboratory, Jining Medical College Affiliated Hospital, Jining Medical College, Jining 272000, Shandong, China

Abstract

Objective(s): The aim of this study was to identify the role of miR-155 in the myocardial ischemia/reperfusion (I/R) injury through targeting hypoxia-inducible factor 1-alpha (HIF-1α).
Materials and Methods: We constructed rat models with myocardial I/R injury and H9C2 cell models with hypoxia/reoxygenation (H/R) damage. Anti-miR-155 and HIF-1α short hairpin RNA (shRNA) were used to treat rats and H9C2 cells to measure infarct area (IA) by TTC staining, determine creatine kinase (CK) and lactate dehydrogenase (LDH) activities by automatic biochemical analyzer, cardiac troponin T (cTnT) and cardiac troponin I (cTnI) levels by ELISA, and detect apoptosis-related proteins by Western blotting. TUNEL staining and flowcytometry were employed to evaluate the apoptosis, JC-1 staining to detect mitochondrial membrane potential (MMP), and MTT assay to determine H9C2 cell viability.
Results: After I/R and H/R, significant elevations were observed in IA, apoptosis, CK, LDH, cTnT, cTnI, and miR-155 levels with reduced HIF-1α. Besides, H/R-induced H9C2 cells presented decreases in MMP and Bcl-2/Bax, but increases in cytosolic/mitochondrial ratio of cytochrome C (Cyt-C) and expressions of cleaved caspase-3 and cleaved caspase-9. However, both rats and H9C2 cells showed an opposite tendency concerning the above after anti-miR-155 treatment. Nevertheless, HIF-1α shRNA effectively reversed protective effects of anti-miR-155 on alleviating I/R- and H/R- induced injury.
Conclusion: Inhibiting miR-155 could reduce myocardial infarct size, suppress I/R-induced cardiomyocyte apoptosis, and maintain the MMP to alleviate I/R-induced injury via specific regulation of HIF-1α.

Keywords

Main Subjects


1. Gu J, Fan Y, Liu X, Zhou L, Cheng J, Cai R, et al. SENP1 protects against myocardial ischaemia/reperfusion injury via a HIF1alpha-dependent pathway. Cardiovasc Res 2014;104:83-92.
2. He B, Xiao J, Ren AJ, Zhang YF, Zhang H, Chen M, et al. Role of miR-1 and miR-133a in myocardial ischemic postconditioning. J Biomed Sci 2011;18:22.
3. Yang J, Chen L, Yang J, Ding J, Li S, Wu H, et al. MicroRNA-22 targeting CBP protects against myocardial ischemia-reperfusion injury through anti-apoptosis in rats. Mol Biol Rep 2014;41:555-561.
4. Hausenloy DJ, Yellon DM. Ischaemic conditioning and reperfusion injury. Nat Rev Cardiol 2016;13:193-209.
5. Kang SM, Lim S, Song H, Chang W, Lee S, Bae SM, et al. Allopurinol modulates reactive oxygen species generation and Ca2+ overload in ischemia-reperfused heart and hypoxia-reoxygenated cardiomyocytes. Eur J Pharmacol 2006;535:212-219.
6. Ottani A, Giuliani D, Galantucci M, Spaccapelo L, Novellino E, Grieco P, et al. Melanocortins counteract inflammatory and apoptotic responses to prolonged myocardial ischemia/reperfusion through a vagus nerve-mediated mechanism. Eur J Pharmacol 2010;637:124-130.
7. Bernink FJ, Timmers L, Beek AM, Diamant M, Roos ST, Van Rossum AC, et al. Progression in attenuating myocardial reperfusion injury: an overview. Int J Cardiol 2014;170:261-269.
8. Ibanez B, Fuster V, Jimenez-Borreguero J, Badimon JJ. Lethal myocardial reperfusion injury: a necessary evil? Int J Cardiol 2011;151:3-11.
9. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136:215-33.
10. Xiao J, Chen YH. MicroRNAs: Novel Regulators of the Heart. J Thorac Dis 2010;2:43-47.
11. Wang GK, Zhu JQ, Zhang JT, Li Q, Li Y, He J, et al. Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J 2010;31:659-666.
12. Ren XP, Wu J, Wang X, Sartor MA, Jones K, Qian J, et al. MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20. Circulation 2009;119:2357-2366.
13. Yu CH, Xu CF, Li YM. Association of MicroRNA-223 expression with hepatic ischemia/reperfusion injury in mice. Dig Dis Sci. 2009;54:2362-2366.
14. Faraoni I, Antonetti FR, Cardone J, Bonmassar E. miR-155 gene: a typical multifunctional microRNA. Biochim Biophys Acta 2009;1792:497-505.
15. Teng G, Papavasiliou FN. Shhh! Silencing by microRNA-155. Philos Trans R Soc Lond B Biol Sci 2009;364:631-637.
16. Heymans S, Corsten MF, Verhesen W, Carai P, van Leeuwen RE, Custers K, et al. Macrophage microRNA-155 promotes cardiac hypertrophy and failure. Circulation 2013;128:1420-1432.
17. Jia QW, Chen ZH, Ding XQ, Liu JY, Ge PC, An FH, et al. Predictive Effects of Circulating miR-221, miR-130a and miR-155 for Coronary Heart Disease: A Multi-Ethnic Study in China. Cell Physiol Biochem 2017;42:808-823.
18. Yang Y, Zhou Y, Cao Z, Tong XZ, Xie HQ, Luo T, et al. miR-155 functions downstream of angiotensin II receptor subtype 1 and calcineurin to regulate cardiac hypertrophy. Exp Ther Med 2016;12:1556-1562.
19. Li X, Kong D, Chen H, Liu S, Hu H, Wu T, et al. miR-155 acts as an anti-inflammatory factor in atherosclerosis-associated foam cell formation by repressing calcium-regulated heat stable protein 1. Sci Rep 2016;6:21789.
20. Wu H, Huang T, Ying L, Han C, Li D, Xu Y, et al. MiR-155 is Involved in renal Ischemia-Reperfusion Injury via Direct Targeting of FoxO3a and Regulating Renal Tubular Cell Pyroptosis. Cell Physiol Biochem 2016;40:1692-1705.
21. Eckle T, Kohler D, Lehmann R, El Kasmi K, Eltzschig HK. Hypoxia-inducible factor-1 is central to cardioprotection: a new paradigm for ischemic preconditioning. Circulation 2008;118:166-175.
22. Bayne K. Revised guide for the care and use of laboratory animals available. American Physiological Society. Physiologist 1996;39:199, 208-211.
23. Seok HY, Chen J, Kataoka M, Huang ZP, Ding J, Yan J, et al. Loss of MicroRNA-155 protects the heart from pathological cardiac hypertrophy. Circ Res 2014;114:1585-95.
24. He W, Huang H, Xie Q, Wang Z, Fan Y, Kong B, et al. MiR-155 knockout in fibroblasts improves cardiac remodeling by targeting tumor protein p53-inducible nuclear protein 1. J Cardiovasc Pharmacol Ther 2016;21:423-435.
25. Amani M, Jeddi S, Ahmadiasl N, Usefzade N, Zaman J. Effect of HEMADO on level of CK-MB and LDH enzymes after ischemia/reperfusion injury in isolated rat heart. Bioimpacts 2013;3:101-104.
26. Zhang X, Liang X, Lin X, Zhang S, Huang Z, Chen C, et al. Mechanism of the protective effect of Yulangsan flavonoid on myocardial ischemia/reperfusion injury in rats. Cell Physiol Biochem 2014;34:1050-1062.
27. Wei L, Sun D, Yin Z, Yuan Y, Hwang A, Zhang Y, et al. A PKC-beta inhibitor protects against cardiac microvascular ischemia reperfusion injury in diabetic rats. Apoptosis. 2010;15:488-498.
28. Xing G, Luo Z, Zhong C, Pan X, Xu X. Influence of miR-155 on cell apoptosis in rats with ischemic stroke: role of the ras homolog enriched in brain (Rheb)/mTOR pathway. Med Sci Monit 2016;22:5141-5153.
29. Jin H, Liu AD, Holmberg L, Zhao M, Chen S, Yang J, et al. The role of sulfur dioxide in the regulation of mitochondrion-related cardiomyocyte apoptosis in rats with isopropylarterenol-induced myocardial injury. Int J Mol Sci. 2013;14:10465-10482.
30. Carraro M, Bernardi P. Calcium and reactive oxygen species in regulation of the mitochondrial permeability transition and of programmed cell death in yeast. Cell Calcium 2016;60:102-107.
31. Wang Y, Li X, Wang X, Lau W, Wang Y, Xing Y, et al. Ginsenoside Rd attenuates myocardial ischemia/reperfusion injury via Akt/GSK-3beta signaling and inhibition of the mitochondria-dependent apoptotic pathway. PLoS One 2013;8:e70956.
32. Murphy KM, Ranganathan V, Farnsworth ML, Kavallaris M, Lock RB. Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells. Cell Death Differ 2000;7:102-111.
33. Dejean LM, Martinez-Caballero S, Kinnally KW. Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis? Cell Death Differ 2006;13:1387-1395.
34. Chen ST, Hsu JR, Hsu PC, Chuang JI. The retina as a novel in vivo model for studying the role of molecules of the Bcl-2 family in relation to MPTP neurotoxicity. Neurochem Res 2003;28:805-814.
35. McDonnell MA, Wang D, Khan SM, Vander Heiden MG, Kelekar A. Caspase-9 is activated in a cytochrome c-independent manner early during TNFalpha-induced apoptosis in murine cells. Cell Death Differ 2003;10:1005-1015.
36. Katoh I, Tomimori Y, Ikawa Y, Kurata S. Dimerization and processing of procaspase-9 by redox stress in mitochondria. J Biol Chem 2004;279:15515-15523.
37. Wang H, Bei Y, Huang P, Zhou Q, Shi J, Sun Q, et al. Inhibition of miR-155 protects against LPS-induced cardiac dysfunction and apoptosis in mice. Mol Ther Nucleic Acids 2016;5:e374.
38. Bruning U, Cerone L, Neufeld Z, Fitzpatrick SF, Cheong A, Scholz CC, et al. MicroRNA-155 promotes resolution of hypoxia-inducible factor 1alpha activity during prolonged hypoxia. Mol Cell Biol 2011;31:4087-4096.
39. Xia X, Kung AL. Preferential binding of HIF-1 to transcriptionally active loci determines cell-type specific response to hypoxia. Genome Biol 2009;10:R113.
40. Madu C, Li L, Lu Y. Selection,  Analysis and improvement of anti-angiogenesis compounds identified by an anti-HIF-1alpha screening and validation system. J Cancer 2016;7:1926-1938.
41. Semenza GL. Hypoxia-inducible factor 1: regulator of mitochondrial metabolism and mediator of ischemic preconditioning. Biochim Biophys Acta 2011;1813:1263-1268.
42. Ong SG, Lee WH, Theodorou L, Kodo K, Lim SY, Shukla DH, et al. HIF-1 reduces ischaemia-reperfusion injury in the heart by targeting the mitochondrial permeability transition pore. Cardiovasc Res 2014;104:24-36.
43. Zhou T, Guo S, Wang S, Li Q, Zhang M. Protective effect of sevoflurane on myocardial ischemia-reperfusion injury in rat hearts and its impact on HIF-1alpha and caspase-3 expression. Exp Ther Med 2017;14:4307-4311.