Abstract
Ischemia–reperfusion (I/R)-induced spinal cord injury can cause apoptotic damage and subsequently act as a blood–spinal cord barrier damage. MicroRNAs (miRNAs) contributed to the process of I/R injury by regulating their target mRNAs. miR-199a-5p is involved in brain and heart I/R injury; however, its function in the spinal cord is not yet completely clarified. In this study, we investigated the role of miR-199a-5p on spinal cord I/R via the endothelin-converting enzyme 1, especially the apoptosis pathway. In the current study, the rat spinal cord I/R injury model was established, and the Basso Beattie Bresnahan scoring, Evans blue staining, HE staining, and TUNEL assay were used to assess the I/R-induced spinal cord injury. The differentially expressed miRNAs were screened using microarray. miR-199a-5p was selected by unsupervised hierarchical clustering analysis. The dual-luciferase reporter assay was used for detecting the regulatory effects of miR-199a-5p on ECE1. In addition, neuron expression was detected by immunostaining assay, while the expressions of p-ERK, ERK, p-JNK, JNK, caspase-9, Bcl-2, and ECE1 were evaluated by Western blot. The results indicated the successful establishment of the I/R-induced spinal cord injury model; the I/R induced the damage to the lower limb motor. Furthermore, 18 differentially expressed miRNAs were detected in the I/R group compared to the sham group, and miR-199a-5p protected the rat spinal cord injury after I/R. Moreover, miR-199a-5p negatively regulated ECE1, and silencing the ECE1 gene also protected the rat spinal cord injury after I/R. miR-199a-5p or silencing of ECE1 also regulated the expressions of caspase-9, Bcl-2, p-JNK, p-ERK, and ECE1 in rat spinal cord injury after I/R. Therefore, we demonstrated that miR-199a-5p might protect the spinal cord against I/R-induced injury by negatively regulating the ECE1, which could aid in developing new therapeutic strategies for I/R-induced spinal cord injury.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Anttila V, Haapanen H, Yannopoulos F, Herajarvi J, Anttila T, Juvonen T (2016) Review of remote ischemic preconditioning: from laboratory studies to clinical trials. Scand Cardiovasc J 50(5–6):355–361. https://doi.org/10.1080/14017431.2016.1233351
Anwar MA, Al Shehabi TS, Eid AH (2016) Inflammogenesis of secondary spinal cord injury. Front Cell Neurosci 10:98. https://doi.org/10.3389/fncel.2016.00098
Arsava EM, Gurer G, Gursoy-Ozdemir Y, Karatas H, Dalkara T (2009) A new model of transient focal cerebral ischemia for inducing selective neuronal necrosis. Brain Res Bull 78(4–5):226–231. https://doi.org/10.1016/j.brainresbull.2008.11.005
Azzarone M, De Troia A, Iazzolino L, Nabulsi B, Tecchio T (2016) Hybrid treatment of acute abdominal aortic thrombosis presenting with paraplegia. Ann Vasc Surg 33:228.e225-228. https://doi.org/10.1016/j.avsg.2015.10.041
Basso DM, Beattie MS, Bresnahan JC (1995) A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12(1):1–21. https://doi.org/10.1089/neu.1995.12.1
Bell MT, Puskas F, Bennett DT, Cleveland JC Jr, Herson PS, Mares JM, Meng X, Weyant MJ, Fullerton DA, Brett Reece T (2015) Clinical indicators of paraplegia underplay universal spinal cord neuronal injury from transient aortic occlusion. Brain Res 1618:55–60. https://doi.org/10.1016/j.brainres.2015.04.053
Bhalala OG, Srikanth M, Kessler JA (2013) The emerging roles of microRNAs in CNS injuries. Nat Rev Neurol 9(6):328–339. https://doi.org/10.1038/nrneurol.2013.67
Borgens RB, Liu-Snyder P (2012) Understanding secondary injury. Q Rev Biol 87(2):89–127
Boyaci MG, Eser O, Kocogullari CU, Karavelioglu E, Tokyol C, Can Y (2014) Neuroprotective effect of alpha-lipoic acid and methylprednisolone on the spinal cord ischemia/reperfusion injury in rabbits. Br J Neurosurg. https://doi.org/10.3109/02688697.2014.954986
Chan YC, Roy S, Huang Y, Khanna S, Sen CK (2012) The microRNA miR-199a-5p down-regulation switches on wound angiogenesis by derepressing the v-ets erythroblastosis virus E26 oncogene homolog 1-matrix metalloproteinase-1 pathway. J Biol Chem 287(49):41032–41043. https://doi.org/10.1074/jbc.M112.413294
Chang CZ, Yen CP, Winadi D, Wu SC, Howng SL, Lin TK, Jeng AY, Kassell NF, Kwan AL (2004) Neuroprotective effect of CGS 26303, an endothelin-converting enzyme inhibitor, on transient middle cerebral artery occlusion in rats. J Cardiovasc Pharmacol 44:S487–S489
Chaturvedi P, Chen NX, O’Neill K, McClintick JN, Moe SM, Janga SC (2015) Differential miRNA expression in cells and matrix vesicles in vascular smooth muscle cells from rats with kidney disease. PLoS ONE 10(6):e0131589. https://doi.org/10.1371/journal.pone.0131589
Cheng SY, Wang SC, Lei M, Wang Z, Xiong K (2018) Regulatory role of calpain in neuronal death. Neural Regen Res 13(3):556–562. https://doi.org/10.4103/1673-5374.228762
Chou AK, Chen TI, Winardi W, Dai MH, Chen SC, Howng SL, Yen CP, Lin TK, Jeng AY, Kwan AL (2007) Functional neuroprotective effect of CGS 26303, a dual ECE inhibitor, on ischemic-reperfusion spinal cord injury in rats. Exp Biol Med 232(2):214–218
El AH, Leptidis S, Dirkx E, Hoeks J, Van BB, Brand K, Mcclellan EA, Poels E, Sluimer JC, Mm VDH (2013) The hypoxia-inducible microRNA cluster miR-199a∼214 targets myocardial PPARδ and impairs mitochondrial fatty acid oxidation. Cell Metab 18(3):341–354
Fang B, Li XQ, Bi B, Tan WF, Liu G, Zhang Y, Ma H (2015) Dexmedetomidine attenuates blood–spinal cord barrier disruption induced by spinal cord ischemia reperfusion injury in rats. Cell Physiol Biochem 36(1):373–383. https://doi.org/10.1159/000430107
Guo D, Liu J, Wang W, Hao F, Sun X, Wu X, Bu P, Zhang Y, Liu Y, Liu F, Zhang Q, Jiang F (2013) Alteration in abundance and compartmentalization of inflammation-related miRNAs in plasma after intracerebral hemorrhage. Stroke 44(6):1739–1742. https://doi.org/10.1161/strokeaha.111.000835
Guo W, Qiu Z, Wang Z, Wang Q, Tan N, Chen T, Chen Z, Huang S, Gu J, Li J, Yao M, Zhao Y, He X (2015) MiR-199a-5p is negatively associated with malignancies and regulates glycolysis and lactate production by targeting hexokinase 2 in liver cancer. Hepatology 62(4):1132–1144. https://doi.org/10.1002/hep.27929
Hashemi Gheinani A, Burkhard FC, Rehrauer H, Aquino Fournier C, Monastyrskaya K (2015) MicroRNA MiR-199a-5p regulates smooth muscle cell proliferation and morphology by targeting WNT2 signaling pathway. J Biol Chem 290(11):7067–7086. https://doi.org/10.1074/jbc.M114.618694
He B, Xiao J, Ren AJ, Zhang YF, Zhang H, Chen M, Xie B, Gao XG, Wang YW (2011) Role of miR-1 and miR-133a in myocardial ischemic postconditioning. J Biomed Sci 18:22. https://doi.org/10.1186/1423-0127-18-22
Henaoui IS, Cauffiez C, Aubert S, Buscot M, Dewaeles E, Copin MC, Marquette CH, Barbry P, Perrais M, Pottier N, Mari B (2013) [miR-199a-5p in idiopathic pulmonary fibrosis]. Med Sci (Paris) 29(5):461–463. https://doi.org/10.1051/medsci/2013295006
Hinkel R, Penzkofer D, Zühlke S, Fischer A, Husada W, Xu QF, Baloch E, Van RE, Zeiher AM, Kupatt C (2013) Inhibition of microRNA-92a protects against ischemia/reperfusion injury in a large-animal model. Circulation 128(10):1066–1075
Hu Y, Deng H, Xu S, Zhang J (2015) MicroRNAs regulate mitochondrial function in cerebral ischemia–reperfusion injury. Int J Mol Sci 16(10):24895–24917. https://doi.org/10.3390/ijms161024895
Ikpeze TC, Mesfin A (2017) Spinal cord injury in the geriatric population: risk factors, treatment options, and long-term management. Geriatr Orthop Surg Rehabil 8(2):115–118. https://doi.org/10.1177/2151458517696680
Jee MK, Jung JS, Choi JI, Jang JA, Kang KS, Im YB, Kang SK (2012) MicroRNA 486 is a potentially novel target for the treatment of spinal cord injury. Brain 135(Pt 4):1237–1252. https://doi.org/10.1093/brain/aws047
Kardes O, Civi S, Tufan K, Oz Oyar E, Omeroglu S, Aykol S (2016) Effects of atorvastatin on experimental spinal cord ischemia–reperfusion injury in rabbits. Turk Neurosurg. https://doi.org/10.5137/1019-5149.jtn.16627-15.2
Keshavarz S, Dehghani GA (2017) Cerebral ischemia/reperfusion injury in the hyperthyroid rat. Iran J Med Sci 42(1):48–56
Khamaisi M, Toukan H, Axelrod JH, Rosenberger C, Skarzinski G, Shina A, Meidan R, Koesters R, Rosen S, Walkinshaw G, Mimura I, Nangaku M, Heyman SN (2015) Endothelin-converting enzyme is a plausible target gene for hypoxia-inducible factor. Kidney Int 87(4):761–770. https://doi.org/10.1038/ki.2014.362
Kilany A, Al-Hashel JY, Rady A (2015) Acute aortic occlusion presenting as flaccid paraplegia. Case Rep Neurol Med 2015:713489. https://doi.org/10.1155/2015/713489
Liang Y, Xu J, Wang Y, Tang JY, Yang SL, Xiang HG, Wu SX, Li XJ (2018) Inhibition of MiRNA-125b decreases cerebral ischemia/reperfusion injury by targeting CK2alpha/NADPH oxidase signaling. Cell Physiol Biochem 45(5):1818–1826. https://doi.org/10.1159/000487873
Liu G, Detloff MR, Miller KN, Santi L, Houle JD (2012) Exercise modulates microRNAs that affect the PTEN/mTOR pathway in rats after spinal cord injury. Experimental neurology 233(1):447–456. https://doi.org/10.1016/j.expneurol.2011.11.018
Liu Y, Zhao J, Zhang W, Gan J, Hu C, Huang G, Zhang Y (2015) lncRNA GAS5 enhances G1 cell cycle arrest via binding to YBX1 to regulate p21 expression in stomach cancer. Scientific reports 5:10159. https://doi.org/10.1038/srep10159
Lorenzo MN, Khan RY, Wang Y, Tai SC, Chan GC, Cheung AH, Marsden PA (2001) Human endothelin converting enzyme-2 (ECE2): characterization of mRNA species and chromosomal localization. Biochim Biophys Acta 1522(1):46–52
Lv HR (2017) lncRNA-Map2k4 sequesters miR-199a to promote FGF1 expression and spinal cord neuron growth. Biochem Biophys Res Commun 490(3):948–954. https://doi.org/10.1016/j.bbrc.2017.06.145
Mathew B, Laden G (2015) Management of severe spinal cord injury following hyperbaric exposure. Diving Hyperb Med 45(3):210
Park HA, Kubicki N, Gnyawali S, Chan YC, Roy S, Khanna S, Sen CK (2011) Natural vitamin E alpha-tocotrienol protects against ischemic stroke by induction of multidrug resistance-associated protein 1. Stroke 42(8):2308–2314. https://doi.org/10.1161/STROKEAHA.110.608547
Sharma HS (2005) Pathophysiology of blood–spinal cord barrier in traumatic injury and repair. Curr Pharm Des 11(11):1353–1389
Sui R, He Z (2014) Polymorphisms of ECE1 may contribute to susceptibility to ischemic stroke in Han Chinese of Northern China. Cell Biochem Biophys 69(2):237–246. https://doi.org/10.1007/s12013-013-9789-z
Sun L, Zhu J, Wu M, Sun H, Zhou C, Fu L, Xu C, Mei C (2015) Inhibition of MiR-199a-5p reduced cell proliferation in autosomal dominant polycystic kidney disease through targeting CDKN1C. Med Sci Monit 21:195–200. https://doi.org/10.12659/MSM.892141
Sun JF, Yang HL, Huang YH, Chen Q, Cao XB, Li DP, Shu HM, Jiang RY (2017) CaSR and calpain contribute to the ischemia reperfusion injury of spinal cord. Neurosci Lett 646:49–55. https://doi.org/10.1016/j.neulet.2017.03.009
Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C, Sharp FR (1990) A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 10(2):290–293. https://doi.org/10.1038/jcbfm.1990.47
Tabayashi K, Takahashi G, Motoyoshi N, Kokubo H, Sakurai M, Oda K, Saiki Y, Iguchi A (2004) Spinal cord protection during most or all of descending thoracic or thoracoabdominal aneurysm repair. Kyobu Geka Jpn J Thoracic Surg 57(4):301–306
Takahashi H, Soma S, Muramatsu M, Oka M, Fukuchi Y (2001) Upregulation of ET-1 and its receptors and remodeling in small pulmonary veins under hypoxic conditions. Am J Physiol Lung Cell Mol Physiol 280(6):L1104-1114. https://doi.org/10.1152/ajplung.2001.280.6.L1104
Ubbink DT, Jacobs MJ (2000) Spinal cord stimulation in critical limb ischemia. A review. Acta Chir Belg 100(2):48–53
Wang XT, Pei DS, Xu J, Guan QH, Sun YF, Liu XM, Zhang GY (2007) Opposing effects of Bad phosphorylation at two distinct sites by Akt1 and JNK1/2 on ischemic brain injury. Cell Signal 19(9):1844–1856. https://doi.org/10.1016/j.cellsig.2007.04.005
Wang W, Yen H, Chen CH, Soni R, Jasani N, Sylvestre G, Reznik SE (2008) The endothelin-converting enzyme-1/endothelin-1 pathway plays a critical role in inflammation-associated premature delivery in a mouse model. Am J Pathol 173(4):1077–1084. https://doi.org/10.2353/ajpath.2008.080257
Wang Y, Liu H, Ma H (2016) Intrathecally transplanting mesenchymal stem cells (MSCs) activates ERK1/2 in spinal cords of ischemia-reperfusion injury rats and improves nerve function. Med Sci Monit 22:1472–1479
Wang Y, Luo J, Wang X, Yang B, Cui L (2017) MicroRNA-199a-5p induced autophagy and inhibits the pathogenesis of ankylosing spondylitis by modulating the mTOR signaling via directly targeting Ras homolog enriched in brain (Rheb). Cell Physiol Biochem 42(6):2481–2491. https://doi.org/10.1159/000480211
Wang Y, Pang QJ, Liu JT, Wu HH, Tao DY (2018) Down-regulated miR-448 relieves spinal cord ischemia/reperfusion injury by up-regulating SIRT1. Braz J Med Biol Res 51(5):e7319. https://doi.org/10.1590/1414-431X20177319
Xu WH, Yao XY, Yu HJ, Huang JW, Cui LY (2012) Downregulation of miR-199a may play a role in 3-nitropropionic acid induced ischemic tolerance in rat brain. Brain Res 1429(1):116
Yamamoto S, Yokomizo Y, Akai T, Chiyoda T, Goto H, Masaki Y (2016) Acute aortic occlusion in a patient with chronic paralysis due to spinal cord injury: a case report. Surg Case Rep 2(1):121. https://doi.org/10.1186/s40792-016-0251-5
Zeng Y, Liu JX, Yan ZP, Yao XH, Liu XH (2015) Potential microRNA biomarkers for acute ischemic stroke. Int J Mol Med 36(6):1639–1647. https://doi.org/10.3892/ijmm.2015.2367
Zeng Y, Ma M, Liu B, Xia J, Xu H, Liu Y, Du X, Hu Z, Yang Q, Zhang L (2016) Association between ECE1 gene polymorphisms and risk of intracerebral haemorrhage. J Int Med Res 44(3):444–452. https://doi.org/10.1177/0300060516635385
Zhao H, Sapolsky RM, Steinberg GK (2006) Phosphoinositide-3-kinase/akt survival signal pathways are implicated in neuronal survival after stroke. Mol Neurobiol 34(3):249–270. https://doi.org/10.1385/mn:34:3:249
Author information
Authors and Affiliations
Contributions
HM and NB conceived and designed the study. NB, BF, HWL, and YHJ performed the experiments. BF, FSC, and ZLW analyzed the data. NB and HM wrote the manuscript. All authors read and approved the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
Ning Bao, Bo Fang, Huangwei Lv, Yanhua Jiang, Fengshou Chen, Zhilin Wang, and Hong Ma declare that they have no financial and personal relationships with people or organizations that can inappropriately influence the current study; there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of the manuscript entitled “Upregulation of miR-199a-5p protects spinal cord against ischemia/reperfusion-induced injury via downregulation of ECE1 in rat.”
Rights and permissions
About this article
Cite this article
Bao, N., Fang, B., Lv, H. et al. Upregulation of miR-199a-5p Protects Spinal Cord Against Ischemia/Reperfusion-Induced Injury via Downregulation of ECE1 in Rat. Cell Mol Neurobiol 38, 1293–1303 (2018). https://doi.org/10.1007/s10571-018-0597-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10571-018-0597-2