INTRODUCTION: Oxidative stress (OS) is a key contributor to secondary damage following spinal cord injury (SCI), leading to neural stem cell (NSC) dysfunction and apoptosis. MicroRNA-20a (miR-20a) is upregulated after SCI and plays a role in regulating apoptosis and survival pathways. This study explores the therapeutic potential of miR-20a inhibition in mitigating OS-induced damage in NSCs. METHODS: Human iPSC-derived NSCs were subjected to oxidative stress by exposure to 100 µM hydrogen peroxide (H2O2) for 2 hours, followed by treatment with a miR-20a inhibitor (100 nM) to attenuate the adverse effects. Metabolic activity was evaluated using the Alamar Blue assay. Apoptotic responses and miR-20a expression levels were assessed via flow cytometry, RT-qPCR, and Western blot analysis. RESULTS: NSCs exposed to OS showed a marked reduction in metabolic activity. However, treatment with a miR-20a inhibitor over 72 h significantly improved cell survival and metabolic activity in a time-dependent manner compared to untreated stressed cells. DISCUSSION: Our findings suggest that miR-20a inhibition mitigates OS-induced cytotoxicity and promotes NSC viability, presenting a potential therapeutic approach for enhancing neural tissue regeneration.

Department of Biochemistry and Microbiology Faculty of Food and Biochemical Technology UCT Prague Prague Czechia

Department of Neuroregeneration Institute of Experimental Medicine CAS Prague Czechia

Department of Neuroscience 2nd Medical Faculty Charles University Prague Czechia

Laboratory of Glial Biology and Omics Technologies Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Vestec Czechia

Zobrazit více v PubMed

Adusumilli V. S., Walker T. L., Overall R. W., Klatt G. M., Zeidan S. A., Zocher S., et al. (2021). ROS dynamics delineate functional states of hippocampal neural stem cells and link to their activity-dependent exit from quiescence. Cell Stem Cell 28, 300–314.e6. doi: 10.1016/J.STEM.2020.10.019, PMID: PubMed DOI PMC

Alizadeh A., Dyck S. M., Karimi-Abdolrezaee S. (2019). Traumatic spinal cord injury: an overview of pathophysiology, models and acute injury mechanisms. Front. Neurol. 10:441408. doi: 10.3389/FNEUR.2019.00282, PMID: PubMed DOI PMC

Alonzi T., Maritano D., Gorgoni B., Rizzuto G., Libert C., Poli V. (2001). Essential role of STAT3 in the control of the acute-phase response as revealed by inducible gene activation in the liver. Mol. Cell. Biol. 21, 1621–1632. doi: 10.1128/MCB.21.5.1621-1632.2001, PMID: PubMed DOI PMC

Andrabi S. A., No S. K., Yu S. W., Wang H., Koh D. W., Sasaki M., et al. (2006). Poly(ADP-ribose) (PAR) polymer is a death signal. Proc. Natl. Acad. Sci. USA 103, 18308–18313. doi: 10.1073/PNAS.0606526103 PubMed DOI PMC

Anjum A., Yazid M. D., Daud M. F., Idris J., Hwei Ng A. M., Naicker A. S., et al. (2020). Spinal cord injury: pathophysiology, multimolecular interactions, and underlying recovery mechanisms. Int. J. Mol. Sci. 21, 1–35. doi: 10.3390/IJMS21207533, PMID: PubMed DOI PMC

Bertrand N., Castro D. S., Guillemot F. (2002). Proneural genes and the specification of neural cell types. Nat. Rev. Neurosci. 3, 517–530. doi: 10.1038/NRN874, PMID: PubMed DOI

Biswas K., Alexander K., Francis M. M. (2022). Reactive oxygen species: angels and demons in the life of a Neuron. Neuro Science 3, 130–145. doi: 10.3390/NEUROSCI3010011, PMID: PubMed DOI PMC

Carraro G., El-Hashash A., Guidolin D., Tiozzo C., Turcatel G., Young B. M., et al. (2009). miR-17 family of microRNAs controls FGF10-mediated embryonic lung epithelial branching morphogenesis through MAPK14 and STAT3 regulation of E-cadherin distribution. Dev. Biol. 333, 238–250. doi: 10.1016/j.ydbio.2009.06.020, PMID: PubMed DOI PMC

Catalá A., Díaz M. (2016). Editorial: Impact of Lipid Peroxidation on the Physiology and Pathophysiology of Cell Membranes. Front. Physiol. 7:423. doi: 10.3389/FPHYS.2016.00423 PubMed DOI PMC

Chan P. H. (2004). Mitochondria and neuronal death/survival signaling pathways in cerebral ischemia. Neurochem. Res. 29, 1943–1949. doi: 10.1007/S11064-004-6869-X, PMID: PubMed DOI

Chan G., Nogalski M. T., Bentz G. L., Smith M. S., Parmater A., Yurochko A. D. (2010). PI3K-dependent upregulation of Mcl-1 by human cytomegalovirus is mediated by epidermal growth factor receptor and inhibits apoptosis in short-lived monocytes. J. Immunol. 184, 3213–3222. doi: 10.4049/JIMMUNOL.0903025, PMID: PubMed DOI PMC

Chen L., Xu B., Liu L., Luo Y., Yin J., Zhou H., et al. (2010). Hydrogen peroxide inhibits mTOR signaling by activation of AMPKα leading to apoptosis of neuronal cells. Lab. Investig. 90, 762–773. doi: 10.1038/labinvest.2010.36, PMID: PubMed DOI PMC

Connor J., Paks C. H., Zwaalt R. F. A., Schroitst A. J. (1992). Bidirectional transbilayer movement of phospholipid analogs in human red blood cells. Evidence for an ATP-dependent and protein-mediated process. J. Biol. Chem. 267, 19412–19417. PubMed

Dello Russo C., Lisi L., Tringali G., Navarra P. (2009). Involvement of mTOR kinase in cytokine-dependent microglial activation and cell proliferation. Biochem. Pharmacol. 78, 1242–1251. doi: 10.1016/J.BCP.2009.06.097, PMID: PubMed DOI

Devaux P. F. (1991). Static and dynamic lipid asymmetry in cell membranes. Biochemistry 30, 1163–1173. doi: 10.1021/BI00219A001, PMID: PubMed DOI

Dimitrijevic M. R., Danner S. M., Mayr W. (2015). Neurocontrol of movement in humans with spinal cord injury. Artif. Organs 39, 823–833. doi: 10.1111/AOR.12614, PMID: PubMed DOI

Epling-Burnette P. K., Liu J. H., Catlett-Falcone R., Turkson J., Oshiro M., Kothapalli R., et al. (2001). Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression. J. Clin. Invest. 107, 351–361. doi: 10.1172/JCI9940 PubMed DOI PMC

Estaquier J., Vallette F., Vayssiere J. L., Mignotte B. (2012). The mitochondrial pathways of apoptosis. Adv. Exp. Med. Biol. 942, 157–183. doi: 10.1007/978-94-007-2869-1_7/COVER PubMed DOI

Fresno Vara J. Á., Casado E., de Castro J., Cejas P., Belda-Iniesta C., González-Barón M. (2004). PI3K/Akt signalling pathway and cancer. Cancer Treat. Rev. 30, 193–204. doi: 10.1016/j.ctrv.2003.07.007, PMID: PubMed DOI

Galluzzi L., Blomgren K., Kroemer G. (2009). Mitochondrial membrane permeabilization in neuronal injury. Nat. Rev. Neurosci. 10:665. doi: 10.1038/nrn2665, PMID: PubMed DOI

Gao X., Qin T., Mao J., Zhang J., Fan S., Lu Y., et al. (2019). PTENP1/miR-20a/PTEN axis contributes to breast cancer progression by regulating PTEN via PI3K/AKT pathway. J Exp Clinic Cancer Res 38:256. doi: 10.1186/S13046-019-1260-6, PMID: PubMed DOI PMC

Gong J., Shen Y., Jiang F., Wang Y., Chu L., Sun J., et al. (2022). MicroRNA-20a promotes non-small cell lung cancer proliferation by upregulating PD-L1 by targeting PTEN. Oncol. Lett. 23:148. doi: 10.3892/OL.2022.13269, PMID: PubMed DOI PMC

Goswami R., Kilkus J., Dawson S., Dawson G. (1999). Overexpression of Akt (protein kinase B) confers protection against apoptosis and prevents formation of ceramide in response to pro-apoptotic stimuli. J. Neurosci. Res. 847–854 [Preprint] doi: 10.1002/(SICI)1097-4547(19990915)57:6 PubMed DOI

Gutierrez J., Ballinger S. W., Darley-Usmar V. M., Landar A. (2006). Free radicals, mitochondria, and oxidized lipids: the emerging role in signal transduction in vascular cells. Circ. Res. 99, 924–932. doi: 10.1161/01.RES.0000248212.86638.E9, PMID: PubMed DOI

Herrmann J. E., Imura T., Song B., Qi J., Ao Y., Nguyen T. K., et al. (2008). STAT3 is a critical regulator of astrogliosis and scar formation after spinal cord injury. J. Neurosci. 28, 7231–7243. doi: 10.1523/JNEUROSCI.1709-08.2008, PMID: PubMed DOI PMC

Islam M. T. (2017). Oxidative stress and mitochondrial dysfunction-linked neurodegenerative disorders. Neurol. Res. 39, 73–82. doi: 10.1080/01616412.2016.1251711, PMID: PubMed DOI

Jee M. K., Jung J. S., Im Y. B., Jung S. J., Kang S. K. (2012). Silencing of miR20a is crucial for Ngn1-mediated neuroprotection in injured spinal cord. Hum. Gene Ther. 23, 508–520. doi: 10.1089/HUM.2011.121, PMID: PubMed DOI

Jung S. Y., Kim D. Y., Yune T. Y., Shin D. H., Baek S. B., Kim C. J. (2014). Treadmill exercise reduces spinal cord injury-induced apoptosis by activating the PI3K/Akt pathway in rats. Exp. Ther. Med. 7, 587–593. doi: 10.3892/ETM.2013.1451, PMID: PubMed DOI PMC

Kamsler A., Segal M. (2007). Control of neuronal plasticity by reactive oxygen species. Antioxid. Redox Signal. 9, 165–167. doi: 10.1089/ARS.2007.9.165, PMID: PubMed DOI

Li H., Zhang X., Qi X., Zhu X., Cheng L. (2019). Icariin inhibits endoplasmic reticulum stress-induced neuronal apoptosis after spinal cord injury through modulating the PI3K/AKT signaling pathway. Int. J. Biol. Sci. 15, 277–286. doi: 10.7150/IJBS.30348, PMID: PubMed DOI PMC

Liang W., Jin W., Ni H., Dai Y., Wang H., Lu T., et al. (2010). Effects of tert-butylhydroquinone on intestinal inflammatory response and apoptosis following traumatic brain injury in mice. Mediat. Inflamm. 2010:564. doi: 10.1155/2010/502564, PMID: PubMed DOI PMC

Liu N. K., Wang X. F., Lu Q. B., Xu X. M. (2009). Altered microRNA expression following traumatic spinal cord injury. Exp. Neurol. 219, 424–429. doi: 10.1016/J.EXPNEUROL.2009.06.015, PMID: PubMed DOI PMC

Liu X. J., Zheng X. P., Zhang R., Guo Y. L., Wang J. H. (2015). Combinatorial effects of miR-20a and miR-29b on neuronal apoptosis induced by spinal cord injury. Int. J. Clin. Exp. Pathol. 8, 3811–3818. PubMed PMC

Lu D. Y., Liou H. C., Tang C. H., Fu W. M. (2006). Hypoxia-induced iNOS expression in microglia is regulated by the PI3-kinase/Akt/mTOR signaling pathway and activation of hypoxia inducible factor-1alpha. Biochem. Pharmacol. 72, 992–1000. doi: 10.1016/J.BCP.2006.06.038, PMID: PubMed DOI

Lv X., Liang J., Wang Z. (2024). MiR-21-5p reduces apoptosis and inflammation in rats with spinal cord injury through PI3K/AKT pathway. Panminerva Med. 66, 256–265. doi: 10.23736/S0031-0808.20.03974-9, PMID: PubMed DOI

Magistretti P. J., Allaman I. (2015). A cellular perspective on brain energy metabolism and functional imaging. Neuron 86, 883–901. doi: 10.1016/J.NEURON.2015.03.035, PMID: PubMed DOI

Maiese K. (2015). Stem cell guidance through the mechanistic target of rapamycin. World J. Stem Cells 7, 999–1009. doi: 10.4252/WJSC.V7.I7.999, PMID: PubMed DOI PMC

Martinez B., Peplow P. (2020). Micrornas as disease progression biomarkers and therapeutic targets in experimental autoimmune encephalomyelitis model of multiple sclerosis. Neural Regen. Res. 15:307. doi: 10.4103/1673-5374.280307, PMID: PubMed DOI PMC

Mormone E., Iorio E. L., Abate L., Rodolfo C. (2023). Sirtuins and redox signaling interplay in neurogenesis, neurodegenerative diseases, and neural cell reprogramming. Front. Neurosci. 17:1073689. doi: 10.3389/FNINS.2023.1073689, PMID: PubMed DOI PMC

Nieto-Diaz M., Esteban F. J., Reigada D., Muñoz-Galdeano T., Yunta M., Caballero-López M., et al. (2014). MicroRNA dysregulation in spinal cord injury: causes, consequences, and therapeutics. Front. Cell. Neurosci. 8:53. doi: 10.3389/FNCEL.2014.00053 PubMed DOI PMC

Polentes J., Jendelova P., Cailleret M., Braun H., Romanyuk N., Tropel P., et al. (2012). Human induced pluripotent stem cells improve stroke outcome and reduce secondary degeneration in the recipient brain. Cell Transplant. 21, 2587–2602. doi: 10.3727/096368912X653228, PMID: PubMed DOI

Riley J. K., Takeda K., Akira S., Schreiber R. D. (1999). Interleukin-10 receptor signaling through the JAK-STAT pathway: requirement for two distinct receptor-derived signals for anti-inflammatory action. J. Biol. Chem. 274, 16513–16521. doi: 10.1074/JBC.274.23.16513, PMID: PubMed DOI

Romanyuk N., Amemori T., Turnovcova K., Prochazka P., Onteniente B., Sykova E., et al. (2015). Beneficial effect of human induced pluripotent stem cell-derived neural precursors in spinal cord injury repair. Cell Transplant. 24, 1781–1797. doi: 10.3727/096368914X684042, PMID: PubMed DOI

Sekiguchi A., Kanno H., Ozawa H., Yamaya S., Itoi E. (2012). Rapamycin promotes autophagy and reduces neural tissue damage and locomotor impairment after spinal cord injury in mice. J. Neurotrauma 29, 946–956. doi: 10.1089/NEU.2011.1919 PubMed DOI

Serrano F., Klann E. (2004). Reactive oxygen species and synaptic plasticity in the aging hippocampus. Ageing Res. Rev. 3, 431–443. doi: 10.1016/J.ARR.2004.05.002, PMID: PubMed DOI

Shin D. Y., Kim G. Y., Lee J. H., Choi B. T., Yoo Y. H., Choi Y. H. (2012). Apoptosis induction of human prostate carcinoma DU145 cells by diallyl disulfide via modulation of JNK and PI3K/AKT signaling pathways. Int. J. Mol. Sci. 13, 14158–14171. doi: 10.3390/IJMS131114158, PMID: PubMed DOI PMC

Silva N. A., Sousa N., Reis R. L., Salgado A. J. (2014). From basics to clinical: a comprehensive review on spinal cord injury. Prog. Neurobiol. 114, 25–57. doi: 10.1016/J.PNEUROBIO.2013.11.002, PMID: PubMed DOI

Stohs S. J., Bagchi D. (1995). Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18, 321–336. doi: 10.1016/0891-5849(94)00159-H, PMID: PubMed DOI

Strickland E. R., Hook M. A., Balaraman S., Huie J. R., Grau J. W., Miranda R. C. (2011). MicroRNA dysregulation following spinal cord contusion: implications for neural plasticity and repair. Neuroscience 186, 146–160. doi: 10.1016/J.NEUROSCIENCE.2011.03.063, PMID: PubMed DOI PMC

Tang Z., Baykal A. T., Gao H., Quezada H. C., Zhang H., Bereczki E., et al. (2014). MTor is a signaling hub in cell survival: a mass-spectrometry-based proteomics investigation. J. Proteome Res. 13, 2433–2444. doi: 10.1021/PR500192G, PMID: PubMed DOI

Taylor R. C., Cullen S. P., Martin S. J. (2008). Apoptosis: controlled demolition at the cellular level. Nat. Rev. Mol. Cell Biol. 9, 231–241. doi: 10.1038/NRM2312, PMID: PubMed DOI

Uyeda A., Muramatsu R. (2020). Molecular mechanisms of central nervous system axonal regeneration and remyelination: A review. Int. J. Mol. Sci. 21, 1–14. doi: 10.3390/ijms21218116 PubMed DOI PMC

Varma A. K., Das A., Wallace G., Barry J., Vertegel A. A., Ray S. K., et al. (2013). Spinal cord injury: a review of current therapy, future treatments, and basic science frontiers. Neurochem. Res. 38, 895–905. doi: 10.1007/S11064-013-0991-6, PMID: PubMed DOI PMC

Wang X., Michaelis E. K. (2010). Selective neuronal vulnerability to oxidative stress in the brain. Front. Aging Neurosci. 2:1224. doi: 10.3389/FNAGI.2010.00012 /BIBTEX PubMed DOI PMC

Wang Y., Yuan Y., Gao Y., Li X., Tian F., Liu F., et al. (2019). MicroRNA-31 regulating apoptosis by mediating the phosphatidylinositol-3 kinase/protein kinase B signaling pathway in treatment of spinal cord injury. Brain Dev. 41, 649–661. doi: 10.1016/J.BRAINDEV.2019.04.010, PMID: PubMed DOI

Wildburger N. C., Lin-Ye A., Baird M. A., Lei D., Bao J. (2009). Neuroprotective effects of blockers for T-type calcium channels. Mol. Neurodegener. 4, 1–8. doi: 10.1186/1750-1326-4-44 PubMed DOI PMC

Yang K., Yao R., Ren L., Wang S., Zhang M. (2021). Euxanthone inhibits traumatic spinal cord injury via anti-oxidative stress and suppression of p38 and PI3K/Akt signaling pathway in a rat model. Transl. Neurosci. 12, 114–126. doi: 10.1515/TNSCI-2021-0012, PMID: PubMed DOI PMC

Yinming C., Benlong W., Hai Z. (2018). Thymoquinone reduces spinal cord injury by inhibiting inflammatory response, oxidative stress and apoptosis via PPAR-γ and PI3K/Akt pathways. Exp. Ther. Med. 15:72. doi: 10.3892/ETM.2018.6072 PubMed DOI PMC

Yu M., Wang Z., Wang D., Aierxi M., Ma Z., Wang Y. (2023). Oxidative stress following spinal cord injury: from molecular mechanisms to therapeutic targets. J. Neurosci. Res. 101, 1538–1554. doi: 10.1002/JNR.25221, PMID: PubMed DOI

Yunta M., Nieto-Díaz M., Esteban F. J., Caballero-López M., Navarro-Ruíz R., Reigada D., et al. (2012). Microrna dysregulation in the spinal cord following traumatic injury. PLoS One 7:534. doi: 10.1371/journal.pone.0034534, PMID: PubMed DOI PMC

Zhao R., Wu X., Bi X. Y., Yang H., Zhang Q. (2022). Baicalin attenuates blood-spinal cord barrier disruption and apoptosis through PI3K/Akt signaling pathway after spinal cord injury. Neural Regen. Res. 17, 1080–1087. doi: 10.4103/1673-5374.324857, PMID: PubMed DOI PMC