• Yongxiang Wang, Jingcheng Wang, Hua Wang, Xinmin Feng, Yuping Tao, Jiandong Yang, and Jun Cai.
• Department of Orthopedics, Clinical Medical College of Yangzhou University, Yangzhou, China; Department of Orthopedics, Northern Jiangsu People's Hospital, Yangzhou, China. Electronic address: wyx918spine@163.com.
• World Neurosurg. 2019 Jun 1; 126: e713-e722.

BackgroundSpinal cord and neuron injury result in loss of muscle function, sensation, or autonomic function in the body. Tet1 produces 5-hydroxymethylcytosin. The conversion was proposed as the initial step of deoxyribonucleic acid demethylation in mammals. However, effects of Tet1 expression and hydroxymethylation status on neuron injury remain unclear. Therefore the current study was designed to explore effects of Tet1 expression and hydroxymethylation status on cell survival and gene expression after neuron injury.MethodsMouse models of spinal cord injury and cell model of neuron injury were created. Animals were sacrificed, and injured spinal cord tissue was harvested. Neuron-like cells were cultured after scratch injury. Hydroxymethylated deoxyribonucleic acid concentration was detected, and Tet1 expression was examined by qPCR. Neuron-like cells were divided into 3 groups: control, injury, and azacytidine + injury (before injury, cells were pretreated with azacytidine) groups. Culture supernatant was collected, and lactate dehydrogenase concentration was detected. Meanwhile, injured neuron-like cells were divided into 3 groups: negative control, Tet1 overexpression, and Tet1 interference. Relative expression of Tet1, BDNF, NTF3, A20, FLIP, HSP70, HSP90, HSP27, and Bcl2 in neuron-like cells was detected by qPCR. In addition, neuron-like cells were divided into 7 groups.ResultsTet1 expression and deoxyribonucleic acid hydroxymethylation increased initially and decreased thereafter after neuron injury in both animal and cell models. Percentages of dead cells increased significantly in neuron-like cells after injury. The percentages of dead cells markedly decreased in injured neuron-like cells that were pretreated with azacytidine. The percentages of dead cells increased markedly in the Tet1 interference group and decreased significantly in the Tet1 overexpression group. Expression of Tet1, BDNF, A20, FLIP, HSP70, HSP90, and Bcl2 decreased significantly after injury. Azacytidine pretreatment in injured neuron-like cells markedly increased expression of Tet1, BDNF, NTF3, A20, FLIP, HSP70, HSP90, HSP27, and Bcl2. Moreover, Tet1 interference significantly decreased the expression of Tet1, BDNF, A20, FLIP, HSP70, and HSP90 in neuron-like cells, whereas Tet1 overexpression markedly increased the expression of Tet1, BDNF, NTF3, A20, FLIP, HSP70, HSP90, HSP27, and Bcl2. BDNF interference significantly increased percentages of dead cells after injury. BDNF interference also markedly decreased the protection of azacytidine and Tet1 overexpression against cell death.ConclusionsTet1 overexpression and demethylation caused by azacytidine protect neurons against cell death after injury by increasing expression of genes involved in cell survival.Copyright © 2019 Elsevier Inc. All rights reserved.

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