Neural Regeneration Research ›› 2018, Vol. 13 ›› Issue (7): 1216-1224.doi: 10.4103/1673-5374.235059

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MicroRNA-219 alleviates glutamate-induced neurotoxicity in cultured hippocampal neurons by targeting calmodulin-dependent protein kinase II gamma

Ting Wang1, 2, Qun Cai3, Wen-Jie Yang4, Hai-Hua Fan4, Jian-Feng Yi4, Feng Xu1   

  1. 1 Department of Emergency, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China;
    2 Department of Emergency, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China;
    3 Department of Pediatrics, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China;
    4 Medical College of Nantong University, Nantong, Jiangsu Province, China
  • Received:2017-12-08 Online:2018-07-15 Published:2018-07-15
  • Contact: Feng Xu, Ph.D., M.D.,suda_xf@163.com.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81101159; the Natural Science Foundation of Jiangsu Province of China, No. BK20151268

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Abstract:

Septic encephalopathy is a frequent complication of sepsis, but there are few studies examining the role of microRNAs (miRs) in its pathogenesis.In this study, a miR-219 mimic was transfected into rat hippocampal neurons to model miR-219 overexpression. A protective effect of miR-219 was observed for glutamate-induced neurotoxicity of rat hippocampal neurons, and an underlying mechanism involving calmodulin-dependent protein kinase II γ (CaMKIIγ) was demonstrated. miR-219 and CaMKIIγ mRNA expression induced by glutamate in hippocampal neurons was determined by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). After neurons were transfected with miR-219 mimic, effects on cell viability and apoptosis were measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. In addition, a luciferase reporter gene system was used to confirm CaMKIIγ as a target gene of miR-219. Western blot assay and rescue experiments were also utilized to detect CaMKIIγ expression and further verify that miR-219 in hippocampal neurons exerted its effect through regulation of CaMKIIγ. MTT assay and qRT-PCR results revealed obvious decreases in cell viability and miR-219 expression after glutamate stimulation, while CaMKIIγ mRNA expression was increased. MTT,flow cytometry, and caspase-3 activity assays showed that miR-219 overexpression could elevate glutamate-induced cell viability, and reduce cell apoptosis and caspase-3 activity. Moreover, luciferase CaMKIIγ-reporter activity was remarkably decreased by co-transfection with miR-219 mimic, and the results of a rescue experiment showed that CaMKIIγ overexpression could reverse the biological effects of miR-219. Collectively, these findings verify that miR-219 expression was decreased in glutamate-induced neurons, CaMKIIγ was a target gene of miR-219, and miR-219 alleviated glutamate-induced neuronal excitotoxicity by negatively controlling CaMKIIγ expression.

Key words: nerve regeneration, brain injury, septic encephalopathy, miR-219, hippocampal neurons, glutamate, excitotoxicity, apoptosis;caspase-3, calmodulin-dependent protein kinase II γ, luciferase reporter gene system, neuroprotection, neural regeneration