miR-196b-5p减轻大鼠脊髓损伤后组织水肿和星形胶质细胞活化机制探讨

Mechanism of miR-196b-5p in alleviating secondary edema and astrocyte activation after spinal cord injury in rats

目的探讨miR-196b-5p靶向水通道蛋白4(AQP4)促进大鼠脊髓损伤(SCI)后组织修复和神经元轴突再生的机制。方法双荧光素酶报告检测miR-196b-5p与AQP4靶向关系;实时定量聚合酶链反应(RT-qPCR)检测SCI后1 d、2 d、3 d、5 d和7 d miR-196b-5p与AQP4变化;RT-qPCR检测假手术(Sham)组、单纯脊髓损伤(SCI)组、agomiR-196b-5p干预(miRNA)组和miR-196b-5p阴性对照(NC)组脊髓miR-196b-5p与AQP4 mRNA相对表达水平,Western blot检测各组脊髓AQP4、胶质纤维酸性蛋白(GFAP)、增殖细胞核抗原(PCNA)和生长相关蛋白43(GAP-43)蛋白相对表达水平,干湿质量法和苏木精-伊红(HE)染色检测脊髓含水量及脊髓空洞大小。结果双荧光素酶报告显示AQP4是miR-196b-5p的靶基因。与Sham组比较,SCI后各时间点miR-196b-5p表达水平降低(P<0.05),而AQP4 mRNA表达升高并在2 d时达到高峰,随后逐渐降低。与Sham组比较,SCI组、miRNA组及NC组AQP4 mRNA及蛋白,GFAP、PCNA和GAP-43蛋白相对表达水平及脊髓含水量升高,而SCI组及NC组miR-196b-5p相对表达水平降低(均P<0.05)。miRNA组AQP4 mRNA,AQP4、GFAP、PCNA蛋白相对表达水平及脊髓含水量较SCI组及NC组降低,脊髓空洞面积减小,而miR-196b-5p和GAP-43相对表达水平升高(均P<0.05)。结论miR-196b-5p通过靶向AQP4抑制SCI后继发性水肿、星形胶质细胞活化增生和胶质瘢痕的形成,最终促进神经元轴突再生和脊髓修复。

Objective To explore the mechanism of miR-196b-5p in promoting tissue repair and neuronal axon regeneration by targeting AQP4 after spinal cord injury(SCI)in rats.Methods The targeting relationship between miR-196b-5p and AQP4 was detected by Dual-Luciferase reporter assay.Changes of miR-196b-5p and AQP4 at 1 d,2 d,3 d,5 d and 7 d after SCI were detected by real time quantitative polymerase reaction(RT-qPCR).The relative expression levels of miR-196b-5p and AQP4 mRNA were detected by RT-qPCR in the Sham group,the SCI group,the miR-196b-5p overexpression(miRNA)group and the miR-196b-5p negative control(NC)group,respectively.The relative expression levels of AQP4,GFAP,PCNA and GAP-43 protein in spinal cord were detect by Western blot assay.Dry-wet weight method and hematoxylin eosin(HE)staining were used to detect water content and size of spinal cord cavity.Results Dual-Luciferase reporter assay showed that AQP4 was the target gene of miR-196b-5p.Compared with the Sham group,the expression of miR-196b-5p decreased at each time point after SCI,while the expression of AQP4 mRNA increased,peaked at 2 days and then decreased gradually(P<0.05).The relative expression levels of AQP4 mRNA,AQP4 protein,GFAP protein,PCNA protein,GAP-43 protein and spinal cord water content increased in the SCI group,the miRNA group and the NC group compared with those of the Sham group(P<0.05),while the relative expression levels of miR-196b-5p decreased in the SCI group and the NC group(P<0.05).The relative expression levels of AQP4 mRNA,AQP4 protein,GFAP protein,PCNA protein and spinal cord water content were lower in the miRNA group than those of the SCI group and the NC group.the area of spinal cord cavity decreased(P<0.05),while the relative expression levels of miR-196b-5p and GAP-43 protein increased(P<0.05).Conclusion miR-196b-5p can inhibit SCI induced secondary edema,astrocyte activation glial scar formation and ultimately promote axon regeneration and spinal cord repair by targeting AQP4.