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

doi:10.11958/20220282

Abstract

Abstract:

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.

Key words: spinal cord injury, edema, aquaporin 4, astrocytes, glial scar, miR-195b-5p

CLC Number:

R651.2

Figures/Tables 7

References 21

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基因名称	引物序列（5'→3'）	产物大小（bp）
AQP4	上游：CACGAAAGATCAGCATCGCC 下游：TTTCCATGAACCGTGGTGACT	216
GAPDH	上游：CTGGAGAAACCTGCCAAGTATG 下游：GGTGGAAGAATGGGAGTTGCT	138

基因名称	引物序列（5'→3'）	产物大小（bp）
AQP4	上游：CACGAAAGATCAGCATCGCC 下游：TTTCCATGAACCGTGGTGACT	216
GAPDH	上游：CTGGAGAAACCTGCCAAGTATG 下游：GGTGGAAGAATGGGAGTTGCT	138

组别	miR-196b-5p	AQP4 mRNA
Sham组	1.00±0.15	1.00±0.18
SCI 1 d组	0.01±0.00^a	13.96±1.30^a
SCI 2 d组	0.02±0.00^a	25.61±2.04^ab
SCI 3 d组	0.07±0.01^a	16.15±0.78^ac
SCI 5 d组	0.11±0.02^a	10.79±1.49^ac
SCI 7 d组	0.11±0.02^a	3.84±0.64^c
F	119.063^＊＊	156.183^＊＊

组别	miR-196b-5p	AQP4 mRNA
Sham组	1.00±0.15	1.00±0.18
SCI 1 d组	0.01±0.00^a	13.96±1.30^a
SCI 2 d组	0.02±0.00^a	25.61±2.04^ab
SCI 3 d组	0.07±0.01^a	16.15±0.78^ac
SCI 5 d组	0.11±0.02^a	10.79±1.49^ac
SCI 7 d组	0.11±0.02^a	3.84±0.64^c
F	119.063^＊＊	156.183^＊＊

组别	miR-196b-5p	AQP4 mRNA	AQP4	脊髓含水量（%）
Sham组	1.00±0.15	1.00±0.31	0.78±0.16	70.17±0.18
SCI组	0.15±0.02^a	24.12±2.26^a	2.67±0.37^a	74.18±0.23^a
miRNA组	0.86±0.03^b	9.66±0.30^ab	1.86±0.26^ab	72.31±0.17^ab
NC组	0.25±0.06^ac	25.53±4.14^ac	2.68±0.30^ac	74.38±0.20^ac
F	80.655^＊＊	74.657^＊＊	30.444^＊＊	299.531^＊＊