MiR-21 inhibitor improves locomotor function recovery by inhibiting IL-6R/JAK-STAT pathway-mediated inflammation after spinal cord injury in model of rat

S.-L. Ning, H. Zhu, J. Shao, Y.-C. Liu, J. Lan, J. Miao

Department of Spine Surgery, Tianjin Hospital, Tianjin, China. 7851256@qq.com

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• Orthopedics

OBJECTIVE: To investigate the function of miRNA-21 and interleukin-6 receptor/Janus Kinase-Signal transducer and activator of transcription (IL-6R/JAK-STAT) pathway in microglia on inflammatory responses after spinal cord injury (SCI).

MATERIALS AND METHODS: This study first detected respectively the protein level of inflammatory factor inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-α) by Western blotting after transfection of miR-21 or administration of miR-21 inhibitor in activated microglia cells of rat in vitro. The quantitative Real-time polymerase chain reaction (qRT-PCR) was utilized to detect the expression of IL-6R under two different interventions. Next, we established a model of spinal cord injury in rat and inspected miR-21 and IL-6R in SCI rat by qRT-PCR. In addition, the protein levels of iNOS and TNF-α in SCI rat were detected by Western blotting. MiR-21 inhibitor was injected into the injured area of SCI rat to delve into the function of miR-21 down-expression on iNOS and TNF-α expression by Western blot as well as the RNA levels of IL-6R, JAK and STAT3 by qRT-PCR. Furthermore, the SCI rat with movement and coordination of hindlimbs was observed by Basso-Beattie-Bresnahan locomotor rating scale (BBB scale) after miR-21 down-expression.

RESULTS: Compared with the microglia transfected with miR-21, the execution of inhibitor in microglia effectively relieved the expression of IL-6R and the breakout of iNOS and TNF-α. Meanwhile, the increase of miR-21 was significantly observed in SCI rat along with significant improvement of inflammatory response-related factors including iNOS and TNF-α. After that, we injected SCI rat with miR-21 inhibitor into the spinal cord injury area and found the inhibition of miR-21 decreased the protein levels of iNOS and TNF-α. Simultaneously, down-expression of miR-21 evidently declined the RNA levels of IL-6R, JAK, and STAT3 in SCI rat. Compared with the sham-operated rat, the movement and coordination of hindlimbs of the SCI group displayed dramatic dysfunction. However, miR-21 down-expression elevated the movement and coordination of hindlimbs of the SCI rat than those of the only injury group.

CONCLUSIONS: Inhibition of miR-21 can promote the recovery of spinal cord injury by down-regulating IL-6R/JAK-STAT signaling pathway and inhibiting inflammation.

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