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| YAN Tingfei,SUN Jingchuan,SUN Chenxi.Effect of miR-195 on microglia autophagy after spinal nerve ligation in rats[J].Chinese Journal of Spine and Spinal Cord,2017,(11):1030-1036. |
| Effect of miR-195 on microglia autophagy after spinal nerve ligation in rats |
| Received:October 30, 2016 Revised:October 27, 2017 |
| English Keywords:MicroRNA-195 Microglia Autophagy associated gene 14 Autophagy Nerve operation Rat |
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| 【Abstract】 Objectives: To explore the effect of microRNA-195(miR-95) on microglia autophagy after spinal nerve ligation and its regulatory mechanism in rats. Methods: Thirty-two adult male SD rats were randomly divided into the spinal nerve ligation model group(SNL group) and the sham operation group(S group), with 16 rats in each group. In SNL group, L5 nerve was ligatured in each rat to prepare spinal nerve ligation model. At each time point of 1d, 3d, 5d, 10d after modeling, 4 rats in each group were executed. Microglia in spinal cord dorsal horn was separated by Percoll gradients centrifugation, real-time PCR was used to detect miR-195 expression. 12 adult male SD rats were sacrificed, microglia in spinal dorsal horn of lumbar enlargements was isolated and divided into two groups, one group with liposome transfection of miR-195 mimics and the other group with transfection of reagent. In the two groups, the expression of autophagic membrane marker protein light chain 3(LC3) was detected. Then microglial cells were divided into three groups, each group contained wild type and mutant autophagy related gene 14(ATG14) 3′ untranslation region(3′UTR) reporter plasmid. HEK293 were transfected by pRL-TK plasmid miR-195 mimics and controls and cultured for 48h. The direct effect of miR-195 target was observed under fluorescent microscope, and the expression of ATG14 protein was detected in three groups. 24 healthy SD rats were sacrificed, microglia in spinal dorsal horn of lumbar enlargements was isolated and divided into three groups, which were transfected with miR-195 mimics, inhibitor and transfection reagent respectively, then cultured for 48h. The expression of ATG14 protein was detected by Western blot in the three groups. Microglial cells were divided into three groups, which were transfected with pEGFP-LC3, pEGFP-LC+miR-195 mimics and pEGFP-LC+miR-195 mimics +pCMV-ATG14, then cultured for 48h. The dot staining of autophagy was observed under fluorescence microscope. Results: The miR-195 expression in microglia of spinal cord dorsal horn increased obviously in the spinal nerve ligation model when compared with S group at the same time, there was significant difference between the two groups(P<0.05). After transfection with miR-195 analog, the expression level of LC-3(0.61±0.07) was significantly lower than that in the control group(1.21±0.08). The fluorescence intensity of wild-type ATG14 3′ UTR plasmid HEK293 cells was significantly lower than that in the control group(0.65±0.04 vs 1.01±0.01), and there was no significant change between the mutant and the control group(0.99±0.03 vs 1.01±0.02). The transfection of miR-195 simulant reduced autophagy level in normal microglia. Luciferase experiments showed ATG14 was the direct target of miR-195, the expression of ATG14 declined after transfection of miR-195 simulant, and rised after transfection of inhibition of miR-19. Conclusions: The miR-195 is highly expressed and inhibited autophagy in microglia of spinal cord dorsal horn in the spinal nerve ligation model. |
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