eISSN: 1896-9151
ISSN: 1734-1922
Archives of Medical Science
Current issue Archive Manuscripts accepted About the journal Special issues Editorial board Abstracting and indexing Subscription Contact Instructions for authors
SCImago Journal & Country Rank
vol. 14
Basic research

MicroRNA-135b-5p prevents oxygen-glucose deprivation and reoxygenation-induced neuronal injury through regulation of the GSK-3β/Nrf2/ARE signaling pathway

Qiang Duan, Wei Sun, Hua Yuan, Xiang Mu

Arch Med Sci 2018; 14, 4: 735–744
Online publish date: 2018/03/28
View full text
Get citation
JabRef, Mendeley
Papers, Reference Manager, RefWorks, Zotero
MicroRNAs (miRNAs) are emerging as critical regulators in the pathological process of cerebral ischemia/reperfusion injury. miRNAs play an important role in regulating neuronal survival. miR-135b-5p has been reported as an important miRNA in regulating cell apoptosis. However, the role of miR-135b-5p in regulating neuronal survival remains poorly understood. Here, we aimed to investigate the role of miR-135b-5p in cerebral ischemia/reperfusion using an in vitro model of oxygen-glucose deprivation and reoxygenation-(OGD/R) induced neuron injury.

Material and methods
miRNA, mRNA and protein expression was detected by real-time quantitative polymerase chain reaction and Western blot. Cell viability was detected by cell counting kit-8 and lactate dehydrogenase assays. Cell apoptosis was detected by caspase-3 activity assay. Oxidative stress was determined using commercial kits. The target of miR-135b-5p was confirmed by dual-luciferase reporter assay.

We found that miR-135b-5p expression was significantly decreased in hippocampal neurons receiving OGD/R treatment. Overexpression of miR-135b-5p markedly alleviated OGD/R-induced cell injury and oxidative stress, whereas suppression of miR-135b-5p showed the opposite effects. We observed that miR-135b-5p directly targeted the 3β-untranslated region of glycogen synthase kinase-3β (GSK-3β). We found that miR-135b-5p negatively regulates the expression of GSK-3β in hippocampal neurons. Moreover, miR-135b-5p overexpression promotes activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling. However, the restoration of GSK-3β expression significantly reversed the protective effects of miR-135b-5p overexpression.

Overall, our results suggest that miR-135b-5p protects neurons against OGD/R-induced injury through downregulation of GSK-3β and promotion of the Nrf2/ARE signaling pathway-mediated antioxidant responses.


GSK-3β, ischemia/reperfusion injury, miR-135b-5p, Nrf2

Feigin VL, Forouzanfar MH, Krishnamurthi R, et al. Global and regional burden of stroke during 1990-2010: findings from the Global Burden of Disease Study 2010. Lancet 2014; 383: 245-54.
Wechsler LR. Intravenous thrombolytic therapy for acute ischemic stroke. N Engl J Med 2011; 364: 2138-46.
Sims NR, Muyderman H. Mitochondria, oxidative metabolism and cell death in stroke. Biochim Biophys Acta 2010; 1802: 80-91.
Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281-97.
Huang Y, Shen XJ, Zou Q, et al. Biological functions of microRNAs: a review. J Physiol Biochem 2011; 67: 129-39.
Di Y, Lei Y, Yu F, et al. MicroRNAs expression and function in cerebral ischemia reperfusion injury. J Mol Neurosci 2014; 53: 242-50.
Ji Q, Gao J, Zheng Y, et al. Inhibition of microRNA-153 protects neurons against ischemia/reperfusion injury in an oxygen-glucose deprivation and reoxygenation cellular model by regulating Nrf2/HO-1 signaling. J Biochem Mol Toxicol 2017 in press.
Wang P, Liang X, Lu Y, et al. MicroRNA-93 downregulation ameliorates cerebral ischemic injury through the Nrf2/HO-1 defense pathway. Neurochem Res 2016; 41: 2627-35.
Seira O, Del Rio JA. Glycogen synthase kinase 3 beta (GSK3beta) at the tip of neuronal development and regeneration. Mol Neurobiol 2014; 49: 931-44.
Leroy K, Brion JP. Developmental expression and localization of glycogen synthase kinase-3beta in rat brain.
J Chem Neuroanat 1999; 16: 279-93.
Woodgett JR. Recent advances in the protein kinase B signaling pathway. Curr Opin Cell Biol 2005; 17: 150-7.
Li Y, Zhu J, Liu Y, et al. Glycogen synthase kinase 3beta influences injury following cerebral ischemia/reperfusion in rats. Int J Biol Sci 2016; 12: 518-31.
Chen X, Liu Y, Zhu J, et al. GSK-3beta downregulates Nrf2 in cultured cortical neurons and in a rat model of cerebral ischemia-reperfusion. Sci Rep 2016; 6: 20196.
Leng Y, Liang MH, Ren M, et al. Synergistic neuroprotective effects of lithium and valproic acid or other histone deacetylase inhibitors in neurons: roles of glycogen synthase kinase-3 inhibition. J Neurosci 2008; 28: 2576-88.
Chan K, Han XD, Kan YW. An important function of Nrf2 in combating oxidative stress: detoxification of acetaminophen. Proc Natl Acad Sci U S A 2001; 98: 4611-6.
Cuadrado A. Structural and functional characterization of Nrf2 degradation by glycogen synthase kinase 3/
beta-TrCP. Free Radic Biol Med 2015; 88: 147-57.
Satoh T, Okamoto SI, Cui J, et al. Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophilic [correction of electrophillic] phase II inducers. Proc Natl Acad Sci U S A 2006; 103: 768-73.
Sandberg M, Patil J, D’Angelo B, et al. NRF2-regulation in brain health and disease: implication of cerebral inflammation. Neuropharmacology 2014; 79: 298-306.
Salazar M, Rojo AI, Velasco D, et al. Glycogen synthase kinase-3beta inhibits the xenobiotic and antioxidant cell response by direct phosphorylation and nuclear exclusion of the transcription factor Nrf2. J Biol Chem 2006; 281: 14841-51.
Pang T, Wang YJ, Gao YX, et al. A novel GSK-3beta inhibitor YQ138 prevents neuronal injury induced by glutamate and brain ischemia through activation of the Nrf2 signaling pathway. Acta Pharmacol Sin 2016; 37: 741-52.
He Y, Wang J, Yung VY, et al. MicroRNA-135b regulates apoptosis and chemoresistance in colorectal cancer by targeting large tumor suppressor kinase 2. Am J Cancer Res 2015; 5: 1382-95.
Li J, Liang H, Bai M, et al. miR-135b promotes cancer progression by targeting transforming growth factor beta receptor II (TGFBR2) in colorectal cancer. PLoS One 2015; 10: e0130194.
Zhang Y, Xing H, Guo S, et al. MicroRNA-135b has a neuroprotective role via targeting of beta-site APP-cleaving enzyme 1. Exp Ther Med 2016; 12: 809-14.
Schaller B, Graf R. Cerebral ischemia and reperfusion: the pathophysiologic concept as a basis for clinical therapy. J Cereb Blood Flow Metab 2004; 24: 351-71.
Li P, Fan JB, Gao Y, et al. miR-135b-5p inhibits LPS-induced TNFalpha production via silencing AMPK phosphatase Ppm1e. Oncotarget 2016; 7: 77978-86.
Song Z, Chen LL, Wang RF, et al. MicroRNA-135b inhibits odontoblast-like differentiation of human dental pulp cells by regulating Smad5 and Smad4. Int Endod J 2017; 50: 685-93.
Xiao S, Yang Z, Lv R, et al. miR-135b contributes to the radioresistance by targeting GSK3beta in human glioblastoma multiforme cells. PLoS One 2014; 9: e108810.
Valeri N, Braconi C, Gasparini P, et al. MicroRNA-135b promotes cancer progression by acting as a downstream effector of oncogenic pathways in colon cancer. Cancer Cell 2014; 25: 469-83.
Fan JB, Ruan JW, Liu W, et al. miR-135b expression downregulates Ppm1e to activate AMPK signaling and protect osteoblastic cells from dexamethasone. Oncotarget 2016; 7: 70613-22.
Hu K, Xie YY, Zhang C, et al. MicroRNA expression profile of the hippocampus in a rat model of temporal lobe epilepsy and miR-34a-targeted neuroprotection against hippocampal neurone cell apoptosis post-status epilepticus. BMC Neurosci 2012; 13: 115.
Xu S, Zhang R, Niu J, et al. Oxidative stress mediated-alterations of the microRNA expression profile in mouse hippocampal neurons. Int J Mol Sci 2012; 13: 16945-60.
Cross DA, Culbert AA, Chalmers KA, et al. Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J Neurochem 2001; 77: 94-102.
Nonaka S, Chuang DM. Neuroprotective effects of chronic lithium on focal cerebral ischemia in rats. Neuroreport 1998; 9: 2081-4.
Wen X, Huang A, Hu J, et al. Neuroprotective effect of astaxanthin against glutamate-induced cytotoxicity in HT22 cells: involvement of the Akt/GSK-3beta pathway. Neuroscience 2015; 303: 558-68.
Li C, Pan Z, Xu T, et al. Puerarin induces the upregulation of glutathione levels and nuclear translocation of Nrf2 through PI3K/Akt/GSK-3beta signaling events in PC12 cells exposed to lead. Neurotoxicol Teratol 2014; 46: 1-9.
Zou Y, Hong B, Fan L, et al. Protective effect of puerarin against beta-amyloid-induced oxidative stress in neuronal cultures from rat hippocampus: involvement of the GSK-3beta/Nrf2 signaling pathway. Free Radic Res 2013; 47: 55-63.
Abdel-Aleem GA, Khaleel EF, Mostafa DG, et al. Neuroprotective effect of resveratrol against brain ischemia reperfusion injury in rats entails reduction of DJ-1 protein expression and activation of PI3K/Akt/GSK3b survival pathway. Arch Physiol Biochem 2016; 122: 200-13.
Sun G, Li Y, Ji Z. Atorvastatin attenuates inflammation and oxidative stress induced by ischemia/reperfusion in rat heart via the Nrf2 transcription factor. Int J Clin Exp Med 2015; 8: 14837-45.
Cheng L, Jin Z, Zhao R, et al. Resveratrol attenuates inflammation and oxidative stress induced by myocardial ischemia-reperfusion injury: role of Nrf2/ARE pathway. Int J Clin Exp Med 2015; 8: 10420-8.
Kim MS, Lee S, Jung N, et al. The vitamin D analogue paricalcitol attenuates hepatic ischemia/reperfusion injury through down-regulation of Toll-like receptor 4 signaling in rats. Arch Med Sci 2017; 13: 459-69.
Qiu HJ, Lu XH, Yang SS, et al. MiR-769 promoted cell proliferation in human melanoma by suppressing GSK3B expression. Biomed Pharmacother 2016; 82: 117-23.
Li M, Chen H, Chen L, et al. miR-709 modulates LPS-induced inflammatory response through targeting GSK-3beta. Int Immunopharmacol 2016; 36: 333-8.
Jiang JJ, Liu CM, Zhang BY, et al. MicroRNA-26a supports mammalian axon regeneration in vivo by suppressing GSK3beta expression. Cell Death Dis 2015; 6: e1865.
Yang X, Wang X, Nie F, et al. miR-135 family members mediate podocyte injury through the activation of Wnt/beta-catenin signaling. Int J Mol Med 2015; 36: 669-77.
Quick links
© 2018 Termedia Sp. z o.o. All rights reserved.
Developed by Bentus.
PayU - płatności internetowe