Cellular and Molecular Neurobiology

, Volume 39, Issue 2, pp 223–240 | Cite as

miR-9 Upregulation Integrates Post-ischemic Neuronal Survival and Regeneration In Vitro

  • Sreekala S. Nampoothiri
  • G. K. RajanikantEmail author
Original Research


The irrefutable change in the expression of brain-enriched microRNAs (miRNAs) following ischemic stroke has promoted the development of radical miRNA-based therapeutics encompassing neuroprotection and neuronal restoration. Our previous report on the systems-level prediction of miR-9 in post-stroke-induced neurogenesis served as a premise to experimentally uncover the functional role of miR-9 in post-ischemic neuronal survival and regeneration. The oxygen-glucose deprivation (OGD) in SH-SY5Y cells significantly reduced miR-9 expression, while miR-9 mimic transfection enhanced post-ischemic neuronal cell viability. The next major objective involved the execution of a drug repositioning strategy to augment miR-9 expression via structure-based screening of Food and Drug Administration (FDA)-approved drugs that bind to Histone Deacetylase 4 (HDAC4), a known miR-9 target. Glucosamine emerged as the top hit and its binding potential to HDAC4 was verified by Molecular Dynamics (MD) Simulation, Drug Affinity Responsive Target Stability (DARTS) assay, and MALDI-TOF MS. It was intriguing that the glucosamine treatment 1-h post-OGD was associated with the increased miR-9 level as well as enhanced neuronal viability. miR-9 mimic or post-OGD glucosamine treatment significantly increased the cellular proliferation (BrdU assay), while the neurite outgrowth assay displayed elongated neurites. The enhanced BCL2 and VEGF parallel with the reduced NFκB1, TNF-α, IL-1β, and iNOS mRNA levels in miR-9 mimic or glucosamine-treated cells further substantiated their post-ischemic neuroprotective and regenerative efficacy. Hence, this study unleashes a potential therapeutic approach that integrates neuronal survival and regeneration via small-molecule-based regulation of miR-9 favoring long-term recovery against ischemic stroke.


MiRNA-9 HDAC4 Glucosamine Ischemic stroke Neuron regeneration Proliferation Neuroprotection Drug repurposing 



Analysis of variance


Adenosine triphosphate


B-cell lymphoma 2




Drug affinity responsive target stability


Dulbecco’s modified Eagle’s medium


Earle’s balanced salt solution


Fetal bovine serum


Food and drug administration


Glyceraldehyde 3-phosphate dehydrogenase


Histone deacetylase-4




Inducible nitric oxide synthase


Lactate dehydrogenase


Matrix-assisted laser desorption/ionization - time-of-flight mass spectrometry


Molecular dynamics


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


Nuclear Factor Kappa B Subunit 1


Oxygen-glucose deprivation


Phosphate-buffered saline


Protein data bank


Propidium iodide


Quantitative real time-polymerase chain reaction


Root-mean-square deviation


Root-mean-square fluctuation


Sodium dodecyl sulfate


Sodium dodecyl sulfate–polyacrylamide gel electrophoresis


Standard error of the mean


Tumor necrosis factor-α


Vascular endothelial growth factor



This study was funded by (a) the Department of Biotechnology, Government of India “Bioinformatics Infrastructure Facility for Biology Teaching through Bioinformatics (BIFBTBI)” (Grant Number: BT/BI/25/001/2006 dated 25/03/2011) and (b) Kerala State Council for Science, Technology and Environment, Science Research Scheme (Grant Number: 018/SRSLS/2014/CSTE).

Author Contributions

SSN and RGK designed experiments; SSN performed experiments, analyzed data, wrote the manuscript; RGK revised the manuscript critically and approved the final version to be submitted.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10571_2018_642_MOESM1_ESM.tif (585 kb)
Supplementary material 1 (TIF 584 KB)


  1. Ashburn TT, Thor KB (2004) Drug repositioning: identifying and developing new uses for existing drugs. Nat Rev Drug Discov 3:673–683CrossRefGoogle Scholar
  2. Bazzoni F, Rossato M, Fabbri M, Gaudiosi D, Mirolo M, Mori L, Tamassia N, Mantovani A, Cassatella MA, Locati M (2009) Induction and regulatory function of miR-9 in human monocytes and neutrophils exposed to proinflammatory signals. Proc Natl Acad Sci USA 106:5282–5287CrossRefGoogle Scholar
  3. Bolger TA, Yao TP (2005) Intracellular trafficking of histone deacetylase 4 regulates neuronal cell death. J Neurosci 25:9544–9553CrossRefGoogle Scholar
  4. Bottomley MJ, Lo Surdo P, Di Giovine P, Cirillo A, Scarpelli R, Ferrigno F, Jones P, Neddermann P, De Francesco R, Steinkühler C, Gallinari P, Carfí A (2008) Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain. J Biol Chem 283:26694–26704CrossRefGoogle Scholar
  5. Chami L, Checler F (2012) BACE1 is at the crossroad of a toxic vicious cycle involving cellular stress and β-amyloid production in Alzheimer’s disease. Mol Neurodegener 7:52CrossRefGoogle Scholar
  6. Chang R, Algird A, Bau C, Rathbone MP, Jiang S (2008) Neuroprotective effects of guanosine on stroke models in vitro and in vivo. Neurosci Lett 431:101–105CrossRefGoogle Scholar
  7. Chen S, Wang M, Yang H, Mao L, He Q, Jin H, Ye ZM, Luo XY, Xia YP, Hu B (2017) LncRNA TUG1 sponges microRNA-9 to promote neurons apoptosis by up-regulated Bcl2l11 under ischemia. Biochem Biophys Res Commun 485:167–173CrossRefGoogle Scholar
  8. Coolen M, Katz S, Bally-Cuif L (2013) miR-9: a versatile regulator of neurogenesis. Front Cell Neurosci 7:220CrossRefGoogle Scholar
  9. Cramer SC (2018) Treatments to promote neural repair after stroke. J Stroke 20:57–70CrossRefGoogle Scholar
  10. Davila JL, Goff LA, Ricupero CL, Camarillo C, Oni EN, Swerdel MR, Toro-Ramos AJ, Li J, Hart RP (2014) A positive feedback mechanism that regulates expression of miR-9 during neurogenesis. PLoS ONE 9:e94348CrossRefGoogle Scholar
  11. Davis CK, Nampoothiri SS, Rajanikant GK (2018) Folic acid exerts post-ischemic neuroprotection in vitro through HIF-1α stabilization. Mol Neurobiol 55(11):8328CrossRefGoogle Scholar
  12. Delaloy C, Liu L, Lee JA, Su H, Shen F, Yang GY, Young WL, Ivey KN, Gao FB (2010) MicroRNA-9 coordinates proliferation and migration of human embryonic stem cell–derived neural progenitors. Cell stem cell 6:323–335CrossRefGoogle Scholar
  13. Dror RO, Dirks RM, Grossman JP, Xu H, Shaw DE (2012) Biomolecular simulation: a computational microscope for molecular biology. Annu Rev Biophys 41:429–452CrossRefGoogle Scholar
  14. Fayaz SM, Rajanikant GK (2014) Ensemble pharmacophore meets ensemble docking: a novel screening strategy for the identification of RIPK1 inhibitors. J Comput Aided Mol Des 28:779–794CrossRefGoogle Scholar
  15. Fordel E, Thijs L, Martinet W, Schrijvers D, Moens L, Dewilde S (2007) Anoxia or oxygen and glucose deprivation in SH-SY5Y cells: a step closer to the unraveling of neuroglobin and cytoglobin functions. Gene 398:114–122CrossRefGoogle Scholar
  16. Fournier NM, Duman RS (2012) Role of vascular endothelial growth factor in adult hippocampal neurogenesis: implications for the pathophysiology and treatment of depression. Behav Brain Res 227:440–449CrossRefGoogle Scholar
  17. Guglielmotto M, Aragno M, Autelli R, Giliberto L, Novo E, Colombatto S, Danni O, Parola M, Smith MA, Perry G, Tamagno E, Tabaton M (2009) The up-regulation of BACE1 mediated by hypoxia and ischemic injury: role of oxidative stress and HIF1alpha. J Neurochem 108:1045–1056CrossRefGoogle Scholar
  18. Gumireddy K, Young DD, Xiong X, Hogenesch JB, Huang Q, Deiters A (2008) Small-molecule inhibitors of microrna miR-21 function. Angew Chem Int Ed Engl 47:7482–7484CrossRefGoogle Scholar
  19. Harari OA, Liao JK (2010) NF-κB and innate immunity in ischemic stroke. Ann N Y Acad Sci 1207:32–40CrossRefGoogle Scholar
  20. Hasan MR, Kim JH, Kim YJ, Kwon KJ, Shin CY, Kim HY, Han SH, Choi DH, Lee J (2013) Effect of HDAC inhibitors on neuroprotection and neurite outgrowth in primary rat cortical neurons following ischemic insult. Neurochem Res 38:1921–1934CrossRefGoogle Scholar
  21. Hawkins M, Barzilai N, Liu R, Hu M, Chen W, Rossetti L (1997) Role of the glucosamine pathway in fat-induced insulin resistance. J Clin Invest 99:2173–2182CrossRefGoogle Scholar
  22. Hwang SY, Shin JH, Hwang JS, Kim SY, Shin JA, Oh ES, Oh S, Kim JB, Lee JK, Han IO (2010) Glucosamine exerts a neuroprotective effect via suppression of inflammation in rat brain ischemia/reperfusion injury. Glia 58:1881–1892CrossRefGoogle Scholar
  23. Iwashita A, Muramatsu Y, Yamazaki T, Muramoto M, Kita Y, Yamazaki S, Mihara K, Moriguchi A, Matsuoka N (2007) Neuroprotective efficacy of the peroxisome proliferator-activated receptor delta-selective agonists in vitro and in vivo. J Pharmacol Exp Ther 320:1087–1096CrossRefGoogle Scholar
  24. Jämsä A, Hasslund K, Cowburn RF, Bäckström A, Vasänge M (2004) The retinoic acid and brain-derived neurotrophic factor differentiated SH-SY5Y cell line as a model for Alzheimer’s disease-like tau phosphorylation. Biochem Biophys Res Commun 319:993–1000CrossRefGoogle Scholar
  25. Jones SW, Watkins G, Le Good N, Roberts S, Murphy CL, Brockbank SM, Needham MR, Read SJ, Newham P (2009) The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13. Osteoarthritis Cartilage 17:464–472CrossRefGoogle Scholar
  26. Khoshnam SE, Winlow W, Farbood Y, Moghaddam HF, Farzaneh M (2017) Emerging roles of microRNAs in ischemic stroke: as possible therapeutic agents. J Stroke 19:166–187CrossRefGoogle Scholar
  27. Kim B, Leventhal PS, Saltiel AR, Feldman EL (1997) Insulin-like growth factor-I-mediated neurite outgrowth in vitro requires mitogen-activated protein kinase activation. J Biol Chem 272:21268–21273CrossRefGoogle Scholar
  28. Krishna A, Biryukov M, Trefois C, Antony PM, Hussong R, Lin J, Heinäniemi M, Glusman G, Köglsberger S, Boyd O, van den Berg BH, Linke D, Huang D, Wang K, Hood L, Tholey A, Schneider R, Galas DJ, Balling R, May P (2014) Systems genomics evaluation of the SH-SY5Y neuroblastoma cell line as a model for Parkinson’s disease. BMC Genom 15:1154CrossRefGoogle Scholar
  29. Laird FM, Cai H, Savonenko AV, Farah MH, He K, Melnikova T, Wen H, Chiang HC, Xu G, Koliatsos VE, Borchelt DR, Price DL, Lee HK, Wong PC (2005) BACE1, a major determinant of selective vulnerability of the brain to amyloid-beta amyloidogenesis, is essential for cognitive, emotional, and synaptic functions. J Neurosci 25:11693–11709CrossRefGoogle Scholar
  30. Lang M-F, Shi Y (2012) Dynamic roles of microRNAs in neurogenesis. Front Neurosci 6:71CrossRefGoogle Scholar
  31. Langley B, Brochier C, Rivieccio MA (2009) Targeting histone deacetylases as a multifaceted approach to treat the diverse outcomes of stroke. Stroke 40:2899–2905CrossRefGoogle Scholar
  32. Lee SY, Lee S, Choi E, Ham O, Lee CY, Lee J, Seo HH, Cha MJ, Mun B, Lee Y, Yoon C, Hwang KC (2018) Small molecule-mediated up-regulation of microRNA targeting a key cell death modulator BNIP3 improves cardiac function following ischemic injury. Sci Rep 8:46973CrossRefGoogle Scholar
  33. Litke C, Bading H, Mauceri D (2018) Histone deacetylase 4 shapes neuronal morphology via a mechanism involving regulation of expression of vascular endothelial growth factor D. J Biol Chem 293:8196–8207CrossRefGoogle Scholar
  34. Liu XS, Chopp M, Zhang RL, Tao T, Wang XL, Kassis H, Hozeska-Solgot A, Zhang L, Chen C, Zhang ZG (2011) MicroRNA profiling in subventricular zone after stroke: MiR-124a regulates proliferation of neural progenitor cells through Notch signaling pathway. PLoS ONE 6:e23461CrossRefGoogle Scholar
  35. Liu XS, Chopp M, Wang XL, Zhang L, Hozeska-Solgot A, Tang T, Kassis H, Zhang RL, Chen C, Xu J, Zhang ZG (2013a) MicroRNA-17-92 cluster mediates the proliferation and survival of neural progenitor cells after stroke. J Biol Chem 288:12478–12488CrossRefGoogle Scholar
  36. Liu XS, Chopp M, Zhang RL, Zhang ZG (2013b) MicroRNAs in cerebral ischemia-induced neurogenesis. J Neuropathol Exp Neurol 72:718–722CrossRefGoogle Scholar
  37. Ma L, Young J, Prabhala H, Pan E, Mestdagh P, Muth D, Teruya-Feldstein J, Reinhardt F, Onder TT, Valastyan S, Westermann F, Speleman F, Vandesompele J, Weinberg RA (2010) miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. Nat Cell Biol 12:247–256CrossRefGoogle Scholar
  38. Nampoothiri SS, Rajanikant GK (2017) Decoding the ubiquitous role of microRNAs in neurogenesis. Mol Neurobiol 54:2003–2011CrossRefGoogle Scholar
  39. Nampoothiri SS, Menon HV, Das D, Krishnamurthy RG (2016) ISCHEMIRs: finding a way through the obstructed cerebral arteries. Curr Drug Targets 17:800–810CrossRefGoogle Scholar
  40. Nampoothiri SS, Fayaz SM, Rajanikant GK (2018) A novel five-node feed-forward loop unravels miRNA-Gene-TF regulatory relationships in ischemic stroke. Mol Neurobiol 55(11):8251CrossRefGoogle Scholar
  41. Ouyang Y-B, Stary CM, Yang G-Y, Giffard R (2013) microRNAs: innovative targets for cerebral ischemia and stroke. Curr Drug Targets 14:90–101CrossRefGoogle Scholar
  42. Pai MY, Lomenick B, Hwang H, Schiestl R, McBride W, Loo JA, Huang J (2015) Drug Affinity Responsive Target Stability (DARTS) for small molecule target identification. Methods Mol Biol 1263:287–298CrossRefGoogle Scholar
  43. Pecot CV, Calin GA, Coleman RL, Lopez-Berestein G, Sood AK (2011) RNA interference in the clinic: challenges and future directions. Nat Rev Cancer 11:59–67CrossRefGoogle Scholar
  44. Qian D, Wei G, Xu C, He Z, Hua J, Li J, Hu Q, Lin S, Gong J, Meng H, Zhou B, Teng H, Song Z (2017) Bone marrow-derived mesenchymal stem cells (BMSCs) repair acute necrotized pancreatitis by secreting microRNA-9 to target the NF-κB1/p50 gene in rats. Sci Rep 7:581CrossRefGoogle Scholar
  45. Radio NM, Mundy WR (2008) Developmental neurotoxicity testing in vitro: models for assessing chemical effects on neurite outgrowth. Neurotoxicology 29:361–376CrossRefGoogle Scholar
  46. Shen Q, Temple S (2009) Fine control: microRNA regulation of adult neurogenesis. Nat Neurosci 12:369–370CrossRefGoogle Scholar
  47. Shi Y, Zhao X, Hsieh J, Wichterle H, Impey S, Banerjee S, Neveu P, Kosik KS (2010) microRNA regulation of neural stem cells and neurogenesis. J Neurosci 30:14931–14936CrossRefGoogle Scholar
  48. Sinoy S, Fayaz SM, Charles KD, Suvanish VK, Kapfhammer JP, Rajanikant GK (2017) Amikacin inhibits miR-497 maturation and exerts post-ischemic neuroprotection. Mol Neurobiol 54:3683–3694CrossRefGoogle Scholar
  49. Traxinger RR, Marshall S (1991) Coordinated regulation of glutamine:-fructose-6-phosphate amidotransferase activity by insulin, glucose, and glutamine. Role of hexosamine biosynthesis in enzyme regulation. J Biol Chem 266:10148–10154Google Scholar
  50. Trazzi S, Fuchs C, Viggiano R, De Franceschi M, Valli E, Jedynak P, Hansen FK, Perini G, Rimondini R, Kurz T, Bartesaghi R, Ciani E (2016) HDAC4: a key factor underlying brain developmental alterations in CDKL5 disorder. Hum Mol Genet 25:3887–3907CrossRefGoogle Scholar
  51. Wang H, Zhang W, Zuo Y, Ding M, Ke C, Yan R, Zhan H, Liu J, Wang J (2015) miR-9 promotes cell proliferation and inhibits apoptosis by targeting LASS2 in bladder cancer. Tumour Biol 36:9631–9640CrossRefGoogle Scholar
  52. Wei N, Xiao L, Xue R, Zhang D, Zhou J, Ren H, Guo S, Xu J (2016) MicroRNA-9 mediates the cell apoptosis by targeting Bcl2l11 in ischemic stroke. Mol Neurobiol 53:6809–6817CrossRefGoogle Scholar
  53. Wishart DS, Knox C, Guo AC, Cheng D, Shrivastava S, Tzur D, Gautam B, Hassanali M (2008) DrugBank: a knowledgebase for drugs, drug actions and drug targets. Nucleic Acids Res 36:D901–D906CrossRefGoogle Scholar
  54. Xiao Z, Li CH, Chan SL, Xu F, Feng L, Wang Y, Jiang JD, Sung JJ, Cheng CH, Chen Y (2014) A small-molecule modulator of the tumor-suppressor miR34a inhibits the growth of hepatocellular carcinoma. Cancer Res 74:6236–6247CrossRefGoogle Scholar
  55. Xicoy H, Wieringa B, Martens GJ (2017) The SH-SY5Y cell line in Parkinson’s disease research: a systematic review. Mol Neurodegener 12:10CrossRefGoogle Scholar
  56. Xie H, Zhao Y, Zhou Y, Liu L, Liu Y, Wang D, Zhang S, Yang M (2017) MiR-9 regulates the expression of BACE1 in dementia induced by chronic brain hypoperfusion in rats. Cell Physiol Biochem 42:1213–1226CrossRefGoogle Scholar
  57. Young DD, Connelly CM, Grohmann C, Deiters A (2010) Small molecule modifiers of microRNA miR-122 function for the treatment of hepatitis C virus infection and hepatocellular carcinoma. J Am Chem Soc 132:7976–7981CrossRefGoogle Scholar
  58. Yuan H, Denton K, Liu L, Li XJ, Benashski S, McCullough L, Li J (2016) Nuclear translocation of histone deacetylase 4 induces neuronal death in stroke. Neurobiol Dis 91:182–193CrossRefGoogle Scholar
  59. Zhao H, Yenari MA, Cheng D, Sapolsky RM, Steinberg GK (2003) Bcl-2 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome c translocation and caspase-3 activity. J Neurochem 85:1026–1036CrossRefGoogle Scholar
  60. Zhao C, Sun G, Li S, Shi Y (2009) A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination. Nat Struct Mol Biol 16:365–371CrossRefGoogle Scholar
  61. Ziemka-Nalecz M, Zalewska T (2014) Neuroprotective effects of histone deacetylase inhibitors in brain ischemia. Acta Neurobiol Exp (Wars) 74:383–395Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of BiotechnologyNational Institute of Technology CalicutCalicutIndia

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