The lncRNA ROR/miR-124-3p/TRAF6 axis regulated the ischaemia reperfusion injury-induced inflammatory response in human cardiac myocytes

  • Ying-Ping Liang
  • Qin Liu
  • Guo-Hai Xu
  • Jing Zhang
  • Yong Chen
  • Fu-Zhou Hua
  • Chang-Qing Deng
  • Yan-Hui HuEmail author


Myocardial ischaemia reperfusion injury (MIRI) is considered the primary cause of death in patients with cardiovascular diseases. Recently, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been found to be involved in the pathogenesis of MIRI. However, whether lncRNA ROR and miR-124-3p play roles in MIRI and the underlying mechanism remain undetermined. HCMs were exposed to hypoxic conditions for 2 h followed by re-oxygenation (H/R) treatment. Expression of miR-124-3p and lncRNA ROR in HCMs was measured by qRT-PCR. TRAF6 expression was evaluated by qRT-PCR and western blotting. ELISA and qRT-PCR were conducted to assess the production of TNF-α, IL-6, and IL-1β. The interaction between miR-124-3p and TRAF6, as well as between miR-124-3p and lncRNA ROR, was verified by dual-luciferase reporter assay. Cell apoptosis was detected by flow cytometry analysis. Our data revealed that miR-124-3p was significantly downregulated, while TRAF6 and lncRNA ROR were upregulated in both MIRI rat model and H/R treated HCMs. Overexpression of miR-124-3p reversed the H/R-induced cell apoptosis and upregulation of TNF-α, IL-6, and IL-1β. Mechanistically, miR-124-3p bound and negatively regulated TRAF6 expression in HCMs. Moreover, TRAF6 overexpression significantly blocked the effects of miR-124-3p mimics on cell apoptosis and inflammatory response of HCMs, which involved the NF-κB pathway. Further analysis showed that lncRNA ROR sponged and negatively regulated miR-124-3p in HCMs. Overexpression of IL-1β was demonstrated to promote H/R induced cell apoptosis in HCMs. In addition, overexpression of ROR further enhanced the H/R-induced inflammation and cell apoptosis through its action on miR-124-3p. The lncRNA ROR/miR-124-3p/TRAF6 axis regulated the H/R-induced cell apoptosis and inflammatory response of HCMs.





Myocardial ischaemia reperfusion injury


Long non-coding RNAs


Micro RNAs


Ischemia reperfusion injury


human cardiac myocytes


TNF receptor associated factor 6


Coronary heart disease


Enzyme Linked Immunosorbent Assay


Quantitative real-time PCR


Wild type




Left anteriordescending coronaryartery



We would like to give our sincere gratitude to the reviewers for their constructive comments.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Anfossi S, Fu X, Nagvekar R, Calin GA (2018) MicroRNAs, regulatory messengers inside and outside Cancer cells. Adv Exp Med Biol 1056:87–108CrossRefGoogle Scholar
  2. Chen X, Sun Y, Cai R, Wang G, Shu X, Pang W (2018) Long noncoding RNA: multiple players in gene expression. BMB Rep 51(6):280–289CrossRefGoogle Scholar
  3. Chen PJ et al (2018) microRNA-874 inhibition targeting STAT3 protects the heart from ischemia-reperfusion injury by attenuating cardiomyocyte apoptosis in a mouse model. J Cell PhysiolGoogle Scholar
  4. Dey BK, Mueller AC, Dutta A (2014) Long non-coding RNAs as emerging regulators of differentiation, development, and disease. Transcription 5(4):e944014CrossRefGoogle Scholar
  5. Frank A, Bonney M, Bonney S, Weitzel L, Koeppen M, Eckle T (2012) Myocardial ischemia reperfusion injury: from basic science to clinical bedside. Semin Cardiothorac Vasc Anesth 16(3):123–132CrossRefGoogle Scholar
  6. Giroud M, Scheideler M (2017) Long Non-Coding RNAs in Metabolic Organs and Energy Homeostasis. Int J Mol Sci 18(12)CrossRefGoogle Scholar
  7. Gong Q, Su G (2017) Roles of miRNAs and long noncoding RNAs in the progression of diabetic retinopathy. Biosci Rep 37(6)Google Scholar
  8. Gonzalez-Montero J et al (2018) Myocardial reperfusion injury and oxidative stress: therapeutic opportunities. World J Cardiol 10(9):74–86CrossRefGoogle Scholar
  9. Hausenloy DJ, Yellon DM (2013) Myocardial ischemia-reperfusion injury: a neglected therapeutic target. J Clin Invest 123(1):92–100CrossRefGoogle Scholar
  10. Huang Y (2018) The novel regulatory role of lncRNA-miRNA-mRNA axis in cardiovascular diseases. J Cell Mol Med 22(12):5768–5775CrossRefGoogle Scholar
  11. Huang S, Ge X, Yu J, Han Z, Yin Z, Li Y, Chen F, Wang H, Zhang J, Lei P (2018) Increased miR-124-3p in microglial exosomes following traumatic brain injury inhibits neuronal inflammation and contributes to neurite outgrowth via their transfer into neurons. FASEB J 32(1):512–528CrossRefGoogle Scholar
  12. Ishida T, Mizushima Si, Azuma S, Kobayashi N, Tojo T, Suzuki K, Aizawa S, Watanabe T, Mosialos G, Kieff E, Yamamoto T, Inoue J (1996) Identification of TRAF6, a novel tumor necrosis factor receptor-associated factor protein that mediates signaling from an amino-terminal domain of the CD40 cytoplasmic region. J Biol Chem 271(46):28745–28748CrossRefGoogle Scholar
  13. Jang YH, Kim JH, Ban C, Ahn K, Cheong JH, Kim HH, Kim JS, Park YH, Kim J, Chun KJ, Lee GH, Kim M, Kim C, Xu Z (2012) Stromal cell derived factor-1 (SDF-1) targeting reperfusion reduces myocardial infarction in isolated rat hearts. Cardiovasc Ther 30(5):264–272CrossRefGoogle Scholar
  14. Jiang W et al (2014) miR-146a ameliorates liver ischemia/reperfusion injury by suppressing IRAK1 and TRAF6. PLoS One 9(7):e101530CrossRefGoogle Scholar
  15. Kong L, Sun M, Jiang Z, Li L, Lu B (2018) MicroRNA-194 inhibits lipopolysaccharide-induced inflammatory response in nucleus Pulposus cells of the intervertebral disc by targeting TNF receptor-associated factor 6 (TRAF6). Med Sci Monit 24:3056–3067CrossRefGoogle Scholar
  16. Lejay A, Fang F, John R, van J, Barr M, Thaveau F, Chakfe N, Geny B, Scholey JW (2016) Ischemia reperfusion injury, ischemic conditioning and diabetes mellitus. J Mol Cell Cardiol 91:11–22CrossRefGoogle Scholar
  17. Li C, Zhao Z, Zhou Z, Liu R (2016) Linc-ROR confers gemcitabine resistance to pancreatic cancer cells via inducing autophagy and modulating the miR-124/PTBP1/PKM2 axis. Cancer Chemother Pharmacol 78(6):1199–1207CrossRefGoogle Scholar
  18. Liu F, Hu H, Zhao J, Zhang Z, Ai X, Tang L, Xie L (2018) miR-124-3p acts as a potential marker and suppresses tumor growth in gastric cancer. Biomed Rep 9(2):147–155PubMedPubMedCentralGoogle Scholar
  19. Loewer S, Cabili MN, Guttman M, Loh YH, Thomas K, Park IH, Garber M, Curran M, onder T, Agarwal S, Manos PD, Datta S, Lander ES, Schlaeger TM, Daley GQ, Rinn JL (2010) Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells. Nat Genet 42(12):1113–1117CrossRefGoogle Scholar
  20. Long HD et al (2018) Reduced hsa-miR-124-3p levels are associated with the poor survival of patients with hepatocellular carcinoma. Mol Biol RepGoogle Scholar
  21. Lu R et al (2018) Prognostic value of lncRNA ROR expression in various cancers: a meta-analysis. Biosci Rep 38(5)Google Scholar
  22. Ong SB, Katwadi K, Kwek XY, Ismail NI, Chinda K, Ong SG, Hausenloy DJ (2018) Non-coding RNAs as therapeutic targets for preventing myocardial ischemia-reperfusion injury. Expert Opin Ther Targets 22(3):247–261CrossRefGoogle Scholar
  23. Qian Z, Zhou S, Zhou Z, Yang X, Que S, Lan J, Qiu Y, Lin Y (2017) miR146b5p suppresses glioblastoma cell resistance to temozolomide through targeting TRAF6. Oncol Rep 38(5):2941–2950CrossRefGoogle Scholar
  24. Quinn JJ, Chang HY (2016) Unique features of long non-coding RNA biogenesis and function. Nat Rev Genet 17(1):47–62CrossRefGoogle Scholar
  25. Rauf A et al (2019) The role of Caspase 1 in ischemia/reperfusion injury of the myocardium. J Cardiovasc PharmacolGoogle Scholar
  26. Sun W et al (2019) Gastrodin ameliorates microvascular reperfusion injury-induced pyroptosis by regulating the NLRP3/caspase-1 pathway. J Physiol BiochemGoogle Scholar
  27. Tan H, Qi J, Fan BY, Zhang J, Su FF, Wang HT (2018) MicroRNA-24-3p attenuates myocardial ischemia/reperfusion injury by suppressing RIPK1 expression in mice. Cell Physiol Biochem 51(1):46–62CrossRefGoogle Scholar
  28. Tang LX, Chen GH, Li H, He P, Zhang Y, Xu XW (2018) Long non-coding RNA OGFRP1 regulates LYPD3 expression by sponging miR-124-3p and promotes non-small cell lung cancer progression. Biochem Biophys Res Commun 505(2):578–585CrossRefGoogle Scholar
  29. Uchida S, Dimmeler S (2015) Long noncoding RNAs in cardiovascular diseases. Circ Res 116(4):737–750CrossRefGoogle Scholar
  30. Walsh MC, Lee J, Choi Y (2015) Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system. Immunol Rev 266(1):72–92CrossRefGoogle Scholar
  31. Wang X, Ha T, Liu L, Zou J, Zhang X, Kalbfleisch J, Gao X, Williams D, Li C (2013) Increased expression of microRNA-146a decreases myocardial ischaemia/reperfusion injury. Cardiovasc Res 97(3):432–442CrossRefGoogle Scholar
  32. Wang X, Ha T, Zou J, Ren D, Liu L, Zhang X, Kalbfleisch J, Gao X, Williams D, Li C (2014) MicroRNA-125b protects against myocardial ischaemia/reperfusion injury via targeting p53-mediated apoptotic signalling and TRAF6. Cardiovasc Res 102(3):385–395CrossRefGoogle Scholar
  33. Wang K, Liu F, Liu CY, An T, Zhang J, Zhou LY, Wang M, Dong YH, Li N, Gao JN, Zhao YF, Li PF (2016) The long noncoding RNA NRF regulates programmed necrosis and myocardial injury during ischemia and reperfusion by targeting miR-873. Cell Death Differ 23(8):1394–1405CrossRefGoogle Scholar
  34. Wang HB, Yang J, Ding JW, Chen LH, Li S, Liu XW, Yang CJ, Fan ZX, Yang J (2016) RNAi-mediated Down-regulation of CD47 protects against ischemia/reperfusion-induced myocardial damage via activation of eNOS in a rat model. Cell Physiol Biochem 40(5):1163–1174CrossRefGoogle Scholar
  35. Wei C, Lei L, Hui H, Tao Z (2019) MicroRNA-124 regulates TRAF6 expression and functions as an independent prognostic factor in colorectal cancer. Oncol Lett 18(1):856–863PubMedPubMedCentralGoogle Scholar
  36. Zhang W, Li Y, Wang P (2018) Long non-coding RNA-ROR aggravates myocardial ischemia/reperfusion injury. Braz J Med Biol Res 51(6):e6555CrossRefGoogle Scholar
  37. Zhang W, Zhao H, Wu Q, Xu W, Xia M (2018) Knockdown of BACE1-AS by siRNA improves memory and learning behaviors in Alzheimer's disease animal model. Exp Ther Med 16(3):2080–2086PubMedPubMedCentralGoogle Scholar
  38. Zhao C et al (2019) Up-regulation of ANXA1 suppresses polymorphonuclear neutrophil infiltration and myeloperoxidase activity by activating STAT3 signaling pathway in rat models of myocardial ischemia reperfusion injury. Cell SignalGoogle Scholar
  39. Zheng C et al (2018) Long noncoding RNA AK12348 is involved in the regulation of myocardial Ischaemia-reperfusion injury by targeting PARP and Caspase-3. Heart Lung Circ 27(5):e51–e58CrossRefGoogle Scholar
  40. Zo RB, Long Z (2018) MiR-124-3p suppresses bladder cancer by targeting DNA methyltransferase 3B. J Cell Physiol 234(1):464–474CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of AnesthesiologySecond Affiliated Hospital of Nanchang UniversityNanchangPeople’s Republic of China
  2. 2.Department of GastroenterologyThe Affiliated Hospital of Jiangxi University of Traditional Chinese MedicineNanchangPeople’s Republic of China

Personalised recommendations