Mechanism of miR-320 in Regulating Biological Characteristics of Ischemic Cerebral Neuron by Mediating Nox2/ROS Pathway

  • Wei Shen
  • Yigao Lu
  • Jun’an Hu
  • Haiwei Le
  • Wei Yu
  • Weihua Xu
  • Wangfang Yu
  • Jiesheng ZhengEmail author


This study aimed to explore the mechanism of miR-320 in regulating biological characteristics of ischemic cerebral neuron by mediating Nox2/ROS pathway. Primary neurons were cultured and grouped: normal group (normal primary neurons), negative control (NC) group (ischemic primary neurons, transfected with negative control plasmid), model group (ischemic primary neurons), miR-320 mimic group (ischemic primary neurons, transfected with miR-320-overexpressed plasmid), Nox2 vector group (ischemic primary neurons, transfected with Nox2-overexpressed plasmid), and miR-320 mimic + Nox2 vector group (ischemic primary neurons, co-transfected with miR-320- and Nox2-overexpressed plasmid). Dual-luciferase reporter assay showed that there was the target relationship between miR-320 and Nox2. miR-320 expression was significantly decreased, and Nox2 expression was significantly increased in the rest groups compared with normal group (both P < 0.05). There was a co-localization of miR-320 and Nox2 in the cytoplasm. Cell proliferation, contents of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX), and mRNA and protein expressions of Ki67, Bcl-2, and c-myc were significantly declined, and apoptosis rate, contents of malondialdehyde (MDA) and reactive oxygen species (ROS), and caspase-3 mRNA and protein expressions were significantly increased in the rest groups compared with normal group (all P < 0.05). miR-320 promoted cell proliferation; increased contents of SOD, CAT, and GSH-PX; and declined apoptosis and contents of MDA and ROS. Moreover, miR-320 could affect the regulation of Nox2/ROS pathway on ischemic cerebral neuron by negatively regulating Nox2 expression. Overexpressed miR-320 affects the proliferation, apoptosis, and oxidative stress injury of ischemic cerebral neuron by inhibiting Nox2/ROS pathway.


miR-320 Nox2/ROS pathway Proliferation Apoptosis Oxidative stress injury 


Funding Information

This work was supported by the Nature Science Foundation Project of Ningbo (No. 2018A610306).

Compliance with ethical standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. Almasi M, Firoozabadi NH, Ghasemi F, Chardoli M (2016) The value of ABCD2F scoring system (ABCD2 combined with atrial fibrillation) to predict 90-day recurrent brain stroke. Neurol Res Int 2016:1–5CrossRefGoogle Scholar
  2. Bisio I, Estatico C, Fedeli A, Lavagetto F, Pastorino M, Randazzo A, Sciarrone A (2018) Brain stroke microwave imaging by means of a Newton-conjugate-gradient method in Lp Banach spaces. IEEE Trans Microw Theory Tech 66:1–15CrossRefGoogle Scholar
  3. Cho RL, Yang CC, Lee IT, Lin CC, Chi PL, Hsiao LD, Yang CM (2016) Lipopolysaccharide induces ICAM-1 expression via a c-Src/NADPH oxidase/ROS-dependent NF-κB pathway in human pulmonary alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 310:L639–L657CrossRefGoogle Scholar
  4. Gheinani AH, Burkhard FC, Monastyrskaya K (2013) Deciphering microRNA code in pain and inflammation: lessons from bladder pain syndrome. Cell Mol Life Sci 70:3773–3789CrossRefGoogle Scholar
  5. Guo Y, Han B, Luo K, Ren Z, Cai L, Sun L (2017) Nox2-ROS-HIF-1α signaling is critical for the inhibitory effect of oleanolic acid on rectal cancer cell proliferation. Biomed Pharmacother 85:733–739CrossRefGoogle Scholar
  6. Hamzei Taj S, Kho W, Riou A, Wiedermann D, Hoehn M (2016) MiRNA-124 induces neuroprotection and functional improvement after focal cerebral ischemia. Biomaterials 91:151–165CrossRefGoogle Scholar
  7. Huang J, Meng Y, Liu Y, Chen Y, Yang H, Chen D, Shi J, Guo Y (2016) MicroRNA-320a regulates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting HOXA10. Cell Physiol Biochem Int J Exp Cell Physiol Biochem Pharmacol 38:40–48CrossRefGoogle Scholar
  8. Jung YS, Lee SW, Park JH, Seo HB, Choi BT, Shin HK (2016) Electroacupuncture preconditioning reduces ROS generation with NOX4 down-regulation and ameliorates blood-brain barrier disruption after ischemic stroke. J Biomed Sci 23:32CrossRefGoogle Scholar
  9. Kim SM, Hur DY, Hong SW, Kim JH (2017a) EBV-encoded EBNA1 regulates cell viability by modulating miR34a-Nox2-ROS signaling in gastric cancer cells. Biochem Biophys Res Commun 494:550–555CrossRefGoogle Scholar
  10. Kim YM, Kim SJ, Tatsunami R, Yamamura H, Fukai T, Ushio-Fukai M (2017b) ROS-induced ROS release orchestrated by Nox4, Nox2 and mitochondria in VEGF signaling and angiogenesis. Am J Physiol Cell Physiol 312:C749–C764CrossRefGoogle Scholar
  11. Lou Z, Wang AP, Duan XM, Hu GH, Zuo ML, Yang ZB (2018) Role of ALK5/SMAD2/3 signaling in the regulation of NOX expression in cerebral ischemia/reperfusion injury. Exp Ther Med 16:1671–1678PubMedPubMedCentralGoogle Scholar
  12. Meng F, Zhang Z, Chen W, Huang G, He A, Hou C, Long Y, Yang Z, Zhang Z, Liao W (2016) MicroRNA-320 regulates matrix metalloproteinase-13 expression in chondrogenesis and interleukin-1β-induced chondrocyte responses. Osteoarthr Cartilage 24:932–941CrossRefGoogle Scholar
  13. Mokhtari T, Akbari M, Malek F, Kashani IR, Rastegar T, Noorbakhsh F, Ghazi-Khansari M, Attari F, Hassanzadeh G (2017) Improvement of memory and learning by intracerebroventricular microinjection of T3 in rat model of ischemic brain stroke mediated by upregulation of BDNF and GDNF in CA1 hippocampal region. Daru J Pharm Sci 25:4CrossRefGoogle Scholar
  14. Qi ZL, Liu YH, Qi SM, Ling LF, Feng ZY, Li Q (2016) Salidroside protects PC12 cells from H2O2-induced apoptosis via suppressing Nox2-ROS-MAPKs signaling pathway. J South Med Univ 37:178–183Google Scholar
  15. Shi C, Zhang Z (2017) MicroRNA-320 suppresses cervical cancer cell viability, migration and invasion via directly targeting FOXM1. Oncol Lett 14:3809–3816CrossRefGoogle Scholar
  16. Song YC, Li WJ, Li LZ (2015) Regulatory effect of miRNA 320a on expression of aquaporin 4 in brain tissue of epileptic rats. Asian Pac J Trop Med 8:807–812CrossRefGoogle Scholar
  17. Sudharani K, Sarma TC, Prasad KS (2016) Brain stroke detection using K-nearest neighbor and minimum mean distance technique. International Conference on ControlGoogle Scholar
  18. Tournier PH, Bonazzoli M, Dolean V, Rapetti F, Hecht F, Nataf F, Aliferis L, El-Kanfoud I, Migliaccio C, de Buhan M, Darbas M, Semenov S, Pichot C (2016) Numerical modeling and high-speed parallel computing: new perspectives on tomographic microwave imaging for brain stroke detection and monitoring. IEEE Antennas & Propagation Magazine PP:1-1Google Scholar
  19. Vishnubalaji R, Hamam R, Yue S, Al-Obeed O, Kassem M, Liu FF, Aldahmash A, Alajez NM (2016) MicroRNA-320 suppresses colorectal cancer by targeting SOX4, FOXM1, and FOXQ1. Oncotarget 7:35789–35802CrossRefGoogle Scholar
  20. Wang J, Shi C, Wang J, Cao L, Zhong L, Wang D (2017) MicroRNA-320a is downregulated in non-small cell lung cancer and suppresses tumor cell growth and invasion by directly targeting insulin-like growth factor 1 receptor. Oncol Lett 13:3247–3252CrossRefGoogle Scholar
  21. Wang X, Chen S, Ni J, Cheng J, Jia J, Zhen X (2018) MiRNA-3473b contributes to neuroinflammation following cerebral ischemia. Cell Death Dis 9:11CrossRefGoogle Scholar
  22. Wu Y, Zhu M, Li D, Zhang Y, Wang Y (2016) Brain stroke localization by using microwave-based signal classification. International Conference on Electromagnetics in Advanced ApplicationsGoogle Scholar
  23. Xu C, Wang X, Gu C, Zhang H, Zhang R, Dong X, Liu C, Hu X, Ji X, Huang S, Chen L (2017a) Celastrol ameliorates Cd-induced neuronal apoptosis by targeting Nox2-derived ROS-dependent PP5-JNK signaling pathway. J Neurochem 141:48–62CrossRefGoogle Scholar
  24. Xu X, Zhang L, Ye X, Hao Q, Zhang T, Cui G, Yu M (2017b) Nrf2/ARE pathway inhibits ROS-induced NLRP3 inflammasome activation in BV2 cells after cerebral ischemia reperfusion. Inflamm Res 67:57–65CrossRefGoogle Scholar
  25. Yang J, Qi J, Xiu B, Yang B, Niu C, Yang H (2019) Reactive oxygen species play a biphasic role in brain ischemia. J Investig Surg 32:97–102CrossRefGoogle Scholar
  26. Yin Q, Lu H, Bai Y, Tian A, Yang Q, Wu J, Yang C, Fan TP, Zhang Y, Zheng X, Zheng X, Li Z (2016) A metabolite of Danshen formulae attenuates cardiac fibrosis induced by isoprenaline, via a Nox2/ROS/p38 pathway. Br J Pharmacol 172:5573–5585CrossRefGoogle Scholar
  27. Zeng LL, He XS, Liu JR, Zheng CB, Wang YT, Yang GY (2016) Lentivirus-mediated overexpression of microRNA-210 improves long-term outcomes after focal cerebral ischemia in mice. CNS Neurosci Ther 22:961–969CrossRefGoogle Scholar
  28. Zhang Y, Wang T, Yang K, Xu J, Wu JM, Liu WL (2016) NADPH oxidase 2 does not contribute to early reperfusion-associated reactive oxygen species generation following transient focal cerebral ischemia. Neural Regen Res 11:1773–1778CrossRefGoogle Scholar
  29. Zhang C, Wang J, Ma X, Wang W, Zhao B, Chen Y, Chen C, Bihl JC (2018) ACE2-EPC-EXs protect ageing ECs against hypoxia/reoxygenation-induced injury through the miR-18a/Nox2/ROS pathway. J Cell Mol Med 22:1873–1882CrossRefGoogle Scholar
  30. Ziaee SM, Tabeshmehr P, Haider KH, Farrokhi M, Shariat A, Amiri A, Hosseini SM (2017) Optimization of time for neural stem cells transplantation for brain stroke in rats. Stem Cell Investig 4:29CrossRefGoogle Scholar

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

Authors and Affiliations

  1. 1.Department of NeurosurgeryBeilun People’s Hospital in NingboNingboChina
  2. 2.Department of NeurosurgeryThe First Affiliated Hospital of Zhejiang UniversityHangzhouChina

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