Effect of miR-412-5p–loaded exosomes in H9c2 cardiomyocytes via the MAPK pathway

Document Type : Original Article


1 Department of Anesthesiology and Pain Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea

2 Department of Energy Information Technology, Fareast University, 76-32, Daehak-gil, Gamgok-myeon, Eumseong-gun, Chungcheongbuk-do 27601, Republic of Korea


Objective(s): MicroRNAs (miRNAs) are small non-coding RNAs that function in all biological processes. Recent findings suggest that exosomes, which are small vesicles abundantly secreted by various cell types, can transport miRNAs to target cells. Here, we elucidated the effect of miRNA-loaded exosomes on lipopolysaccharide (LPS)-induced inflammation in H9c2 cardiomyocytes.
Materials and Methods: Exosomes were isolated from mesenchymal stem cells (MSC) and loaded with miR-412-5p. Additionally, the effect of the miR-412-5p-loaded exosomes on LPS-induced inflammation in H9c2 cardiomyocytes was evaluated by assessing the levels of nitric oxide (NO), reactive oxygen species (ROS), and prostaglandin E2 (PGE2). The expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), inflammatory cytokines, and mitogen-activated protein kinase (MAPK) signaling factors was evaluated using reverse transcription-quantitative PCR and western blotting.
Results: miR-412-5p-loaded exosomes inhibited LPS-induced secretion of inflammatory mediators (NO, PGE2, and ROS), pro-inflammatory cytokines (IL-1β and IL-6), and COX-2 and iNOS expression. Additionally, miR-412-5p-loaded exosomes significantly decreased the expression of MAPK signaling molecules, including p-extracellular signal-regulated kinase (ERK), p-p38, and p-Jun kinase (JNK), in H9c2 cardiomyocytes.
Conclusion: These findings showed that miR-412-5p-loaded exosomes ameliorated LPS-induced inflammation in H9c2 cardiomyocytes by inhibiting COX-2 and iNOS expression, inflammatory mediators, and pro-inflammatory cytokines via the MAPK pathway. The findings indicate that miR-412-5p-loaded exosomes may be effective for the prevention of myocardial injury.


Main Subjects

1. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281-297.
2. Bartel DP. MicroRNAs: Target recognition and regulatory functions. Cell 2009; 136: 215-233. 
3. Jiang Q, Wang Y, Hao Y, Juan L, Teng M, Zhang X, et al. miR2Disease: A manually curated database for microRNA deregulation in human disease. Nucleic Acids Res 2009; 37: D98-104.
4. Mathivanan S, Ji H, Simpson RJ. Exosomes: Extracellular organelles important in intercellular communication. J Proteomics 2010; 73: 1907-1920.
5. Hu G, Drescher KM, Chen XM. Exosomal miRNAs: Biological properties and therapeutic potential. Front Genet 2012; 3: 56-64.
6. Yeo RW, Lai RC, Zhang B, Tan SS, Yin Y, Teh BJ, et al. Mesenchymal stem cell: An efficient mass producer of exosomes for drug delivery. Adv Drug Deliv Rev 2013; 65: 336-341.
7. Lai RC, Arslan F, Lee MM, Sze NS, Choo A, Chen TS, et al. Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res 2010; 4: 214-222. 
8. Hu GW, Li Q, Niu X, Hu B, Liu J, Zhou SM, et al. Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice. Stem Cell Res 2015; 6: 1-5. 
9. Zhang B, Yin Y, Lai RC, Tan SS, Choo AB, Lim SK. Mesenchymal stem cells secrete immunologically active exosomes. Stem Cells Dev 2014; 23: 1233-1244. 
10. Andaloussi SE, Lakhal S, Mäger I, Wood MJ. Exosomes for targeted siRNA delivery across biological barriers. Adv Drug Deliv Rev 2013; 65: 391-397.
11. Amiri A, Bagherifar R, Dezfouli EA, Kiaie SH, Jafari R, Ramezani R. Exosomes as bio-inspired nanocarriers for RNA delivery: Preparation and applications. J Transl Med. 2022; 20:  125-140
12. Fu S, Wang Y, Xiaa X, Zheng J. Exosome engineering: Current progress in cargo loading and targeted delivery. NanoImpact 2020; 20: 1000261.
13. Moon B, Chang S. Exosome as a delivery vehicle for cancer therapy. Cells 2022; 11: 316-330
14. Fang Z, Zhang X, Huang H, Wu J. Exosome based miRNA delivery strategy for disease treatment. Chin Chem Lett 2021; 33: 1693-1704. 
15. Ohno SI, Takanashi M, Sudo K, Ueda S, Ishikawa A, Matsuyama N, et al. Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol Ther 2013; 21: 185-191.
16. Lou G, Song X, Yang F, Wu S, Wang J, Chen Z, et al. Exosomes derived from miR-122-modified adipose tissue-derived MSCs increase chemosensitivity of hepatocellular carcinoma. J Hematol Oncol 2015; 8: 1–11.
17. Katakowski M, Buller B, Zheng X, Lu Y, Rogers T, Osobamiro O, et al. Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth. Cancer Lett 2013; 335: 201-204.
18. Tupchong K, Koyfman A, Foran M. Sepsis, severe sepsis, and septic shock: A review of the literature. Afr J Emerg Med 2015; 5: 127-135.
19. Habimana R, Choi I, Cho HJ, Kim D, Lee K, Jeong I. Sepsis-induced cardiac dysfunction: A review of pathophysiology. Acute Crit Care 2020; 35: 57-66
20. Shvilkina T, Shapiro N. Sepsis-induced myocardial dysfunction: Heterogeneity of functional effects and clinical significance. Front Cardiovasc Med 2023; 10: 1200441-1200448.
21. L’Heureux M, Sternberg M, Brath L, Turlington J, Kashiouris MG. Sepsis-induced cardiomyopathy: A comprehensive review. Curr Cardiol Rep 2020; 22: 35-46.
22. Court O, Kumar A, Parrillo JE, Kumar A. Clinical review: Myocardial depression in sepsis and septic shock. Crit Care 2002; 6: 500-508.
23. Frazier WJ, Xue J, Luce WA, Liu Y. MAPK signaling drives inflammation in LPS-stimulated cardiomyocytes: The route of crosstalk to G-protein-coupled receptors. PloS One 2012; 7: e50071-50080.
24. Diana LM, Hickson‐Bick, Jones C, Buja LM. Stimulation of mitochondrial biogenesis and autophagy by lipopolysaccharide in the neonatal rat cardiomyocyte protects against programmed cell death. J Mol Cell Cardiol 2008; 44: 411-418.
25. Knapp S, Branger J, Van der Poll T. Advances in research of the inflammatory response: The importance of Toll-like receptors. Wien Med Wochenschr 2002; 152: 552-554.
26. Lu YC, Yeh WC, Ohashi PS. LPS/TLR4 signal transduction pathway. Cytokine 2008; 42: 145-151.
27. Song D, Liu J, Wang F, Li X, Liu M, Zhang Z, et al. Procyanidin B2 inhibits lipopolysaccharide-induced apoptosis by suppressing the Bcl-2/Bax and NF-κB signalling pathways in human umbilical vein endothelial cells. Mol Med Rep 2021; 23: 267-280 
28. Ock J, Kim S, Suk K. Anti-inflammatory effects of a fluorovinyloxyacetamide compound KT-15087 in microglia cells. Pharmacol Res 2009; 59: 414-422.
29. Lee JW, Bae CJ, Choi YJ, Kim SI, Kim NH, Lee HJ, et al. 3, 4, 5-Trihydroxycinnamic acid inhibits LPS-induced iNOS expression by suppressing NF-κB activation in BV2 microglial cells. Korean J Physiol Pharmacol 2012; 16: 107-112. 
30. Shindo T, Ikeda U, Ohkawa F, Kawahara Y, Yokoyama M, Shimada K. Nitric oxide synthesis in cardiac myocytes and fibroblasts by inflammatory cytokines. Cardiovasc Res 1995; 29: 813-819.
31. Haudek SB, Spencer E, Bryant DD, White DJ, Maass D, Horton JW, et al. Overexpression of cardiac I-κBα prevents endotoxin-induced myocardial dysfunction. Am J Physiol Heart Circ Physiol 2001; 280: H962-968.
32. Chen W, Zhong Y, Feng N, Guo Z, Wang S, Xing D. New horizons in the roles and associations of COX-2 and novel natural inhibitors in cardiovascular diseases. Mol Med 2021; 27: 123-3033.  
33. Wang MJ, Jeng KC, Kuo JS, Chen HL, Huang HY, Chen WF, et al. c-Jun N-terminal kinase and, to a lesser extent, p38 mitogenactivated protein kinase regulate inducible nitric oxide synthase expression in hyaluronan fragments-stimulated BV-2 microglia. J Neuroimmunol 2004; 146: 50-62.
34. Yang D, Li Z, Gao G, Li X, Liao Z, Wang Y, et al. Combined analysis of surface protein profile and microRNA expression profile of exosomes derived from brain microvascular endothelial cells in early cerebral ischemia. ACS Omega 2021; 6: 22410-22421.
35. Gomez I, Foudi N, Longrois D, Norel X. The role of prostaglandin E2 in human vascular inflammation. Prostaglandins Leukot Essent Fat Acids 2013; 89: 55-63. 
36. Baumgarten G, Knuefermann P, Schuhmacher G, Vervölgyi V, von Rappard J, Dreiner U, et al. Toll-like receptor 4, nitric oxide, and myocardial depression in endotoxemia. Shock 2006; 25: 43-49.
37. Khan S, Andrews KL., Chin-Dusting JP. Cyclo-oxygenase (COX) inhibitors and cardiovascular risk: Are non-steroidal anti-inflammatory drugs really anti-inflammatory? Int J Mol Sci 2019; 20: 4262-4279.
38. Kaminska B. MAPK signalling pathways as molecular targets for anti-inflammatory therapy--from molecular mechanisms to therapeutic benefits. Biochim Biophys Acta 2005; 1754: 253-262
39. Peti W, Page R. Molecular basis of MAP kinase regulation. Protein Sci 2013; 22: 1698-1710.
40. Murakami A, Ohigashi H. Targeting NOX, INOS and COX-2 in inflammatory cells: chemoprevention using food phytochemicals. Int J Cancer 2007; 121: 2357-2363.
41. Buchholz KR, Stephens RS. The extracellular signal-regulated kinase/mitogen-activated protein kinase pathway induces the inflammatory factor interleukin-8 following Chlamydia trachomatis infection. Infect Immun 2007; 75: 5924-5929.
42. Börsch-Haubold AG, Pasquet S, Watson SP. Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059. SB 203580 also inhibits thromboxane synthase. J Biol Chem 1998; 273: 28766-28772. 
43. Wang C, Lu H, Luo C, Song C, Wang Q, Peng Y, et al. miR-412-5p targets Xpo1 to regulate angiogenesis in hemorrhoid tissue. Gene 2019; 705: 167-176.
44. Ashare A, Powers LS, Butler NS, Doerschug KC, Monick MM, Hunninghake GW. Anti-inflammatory response is associated with mortality and severity of infection in sepsis. Am J Physiol Lung Cell Mol Physiol 2005; 288: L633-640.
45. Gómez MI, Lee A, Reddy B, Muir A, Soong G, Pitt A, et al. Staphylococcus aureus protein A induces airway epithelial inflammatory responses by activating TNFR1. Nat Med 2004; 10: 842-848.