Expression of miR-210 mediated by adeno-associated virus performed neuroprotective effects on a rat model of acute spinal cord injury.

Acute spinal cord injuries (ASCI) are common neural disorders in traumatology medicine. MicroRNA-210 (miR-210) plays a crucial role in cell survival, endothelial cell migration and cell regeneration. This paper is aim to validate the pathophysiological function of miR-210 on ASCI. We built a rat model of ASCI and utilized an adeno-associated virus (rAAV)-expressing miR-210 for stable over-expression of miR-210. We tested in vivo miR-210 gain of function on ASCI by microinjected rAAV-miR-210 into the rat spinal cord. We further screened the targeting genes of miR-210 by PCR array and detected related signal proteins by Western Blot and qPCR. Over-expression of miR-210 protected neurons while neurologic function scores were improved. We further identified less TUNEL-positive cells, few features of apoptosis under electron microscopy, decreased activities of caspase-3 and 8 and increased vessel count in the spinal cord from rAAV-miR-210 group. We also found rAAV-miR-210 promoted expression of angiogenesis and metastasis-related protein (VEGF and Glut1) and regulated serum levels of inflammation-related cytokines. PCR screen array showed PTP1B, target of miR-210, was significantly down-regulated and Akt phosphorylation was significantly increased in rAAV-miR-210 group. The current data suggest that over-expression of miR-210 may target PTP1B and plays a neuroprotective role on rats after ASCI.

[Construction and screening of neurite outgrowth inhibitory 66 eukaryotic expression vectors].

OBJECTIVE: To construct and screen neurite outgrowth inhibitory 66-small interfering RNA (nogo66-siRNA) eukaryotic expression vectors of effective interference, so as to lay a foundation for further reconstruction of related viral vector. METHODS: The nogo66-siRNA fragments were designed and cloned into pGenesil-1.1, 4 plasmids of pGenesil-nogo66-siRNA-1, pGenesil-nogo66-siRNA-2, pGenesil-nogo66-siRNA-hk, and pGenesil-nogo66-siRNA-kb were obtained, sequenced and identified, then were transfected into C6 cell line. The transfection efficiency was measured by fluorescence microscope. RT-PCR and Western blot were used to detect the expression of nogo gene and select the plasmid of effective interference. RESULTS: DNA sequencing results showed interference sequences were correct. The bands of 800 bp and 4.3 kb were detected when pGenesil-nogo66-siRNAs were digested by Kpn I/Xho I. The expression of green fluorescent protein could be detected under fluorescence microscope, and the transfection efficiency was about 73%. RT-PCR and Western blot results showed that compared to non-transfected cells, the transfection of pGenesil-nogo66-siRNA-1 made the expression of nogo gene decline 22% and the expression of nogo protein decline 73%; the transfection of pGenesil-nogo66-siRNA-2 made the expression of nogo gene decline 28% and the expression of nogo protein decline 78%; the differences were significant (P < 0.05); and the transfection of pGenesil-nogo66-siRNA-hk and pGenesil-nogo66-siRNA-kb did not make the expressions of nogo gene and nogo protein decrease significantly (P > 0.05). CONCLUSION: Nogo66-siRNA eukaryotic expression vector is successfully constructed, it lays an experimental foundation for repair of spinal cord injury.