Electro-acupuncture promotes the proliferation of neural stem cells and the survival of neurons by downregulating miR-449a in rat with spinal cord injury

Abstract

The aim of this study is to investigate the mechanism of electro-acupuncture (EA) on the recovery of injured spinal cord. Rats were randomly divided into normal control, sham-operated, SCI, SCI+EA group and T10 segment spinal cord injury (SCI) rat model was established by the modified Allen’s method. After 7 days, the mRNA and protein expression of Nestin, neuron specific nuclear protein (NeuN) and calcitonin gene related peptide (CGRP) were detected by real time RT-PCR, Western blot and immunohistochemistry respectively. The protein expression of cleaved caspase 3, Bax, Bcl-2, TNF-α and IL-1β were also detected by Western blot. MicroRNA 449a (miR- 449a) expression was also compared. Further, 12 SCI rats were randomly divided into EA and miR subgroups (EA + miR-449a agomir injection). The expression of Nestin, NeuN, CGRP, cleaved caspase 3, Bax, Bcl-2, TNF-α, IL-1β and miR-449a was compared. The direct interaction of miR-449a and CGRP mRNA was assessed by dual luciferase reporter assay. At day 7, compared with sham-operated group, miR-449a expression in SCI group was significantly increased (P < 0.05), and NeuN and CGRP mRNA and protein expression was markedly decreased (P < 0.05), but protein levels of Nestin, cleaved caspase 3, TNF-α, IL-1β and the ratio of Bax/Bcl-2 in SCI group were significantly increased (P < 0.05). The EA treatment significantly reduced miR-449a level and cleaved caspase 3, TNF-α, IL-1β level and the ratio of Bax/Bcl-2 (P < 0.01), but substantially increased Nestin, NeuN and CGRP expression (P < 0.05 or 0.01). High level of iR-449a in miR subgroup was accompanied by decreased expression of Nestin, NeuN and CGRP and increased expression of cleaved caspase 3, TNF-α, IL-1β and elevation of the ratio of Bax/Bcl-2 (P < 0.05), suggesting miR-449a inhibits the effects of EA on NSCs and neurons. Lucif- erase reporter assay showed that miR-449a bound to the 3’ UTR of CGRP, and thereby regulated CGRP expres- sion. In conclusion, EA promotes proliferation of neural stem cells and the survival of neurons by downregulation of miR-449a expression.