miRNA-221 Regulates Spinal Cord Injury-Induced Inflammatory Response through Targeting TNF- α Expression

Abstract

Objectives: To investigate the roles of miR-221 in spinal cord injury (SCI) as well as the underlying mechanism.

Methods: A mouse model of SCI was generated and used to examine dynamic changes in grip strength of the mouse upper and lower limbs. The expression of miR-221 and tumor necrosis factor-α (TNF-α) was detected by RT-qPCR and Western blot. Levels of inflammation and oxidative stress in microglia cells of the injured mice overexpressing miR-221 were then measured by ELISA. Bioinformatics analysis and dual-luciferase reporter assay were conducted to identify the miR-221 target.

Results: We successfully constructed SCI mouse model. The results of qRT-PCR showed that miR-221 was gradually upregulated in the spinal cord tissue of mice in the SCI group with the prolonged injury time. At the same time, the mRNA and protein of TNF-α gradually decreased. We further confirmed through cell experiments that the inflammatory factors TNF-α and IL-6, as well as iNOS and eROS, were upregulated in spinal cord microglia cells of SCI mice, and upregulation of miR-122 can inhibit their expression. Finally, the luciferase reporter experiment confirmed that miR-122 targeted TNF-α.

Conclusions: We present evidence that miR-221 promotes functional recovery of the injured spinal cord through targeting TNF-α, while alleviating inflammatory response and oxidative stress.

Figure 1

SCI mouse model was successfully established. (a) Grip strength of mice in the pair forepaws. (b) Observation of morphology of spinal cord tissues in the sham and SCI group detected by HE staining.

Figure 2

Dynamic changes of miR-221 and TNF-α expression. (a) A significant time-dependent increase in miR-221 expression during the injury. (b–d) The time-dependent decrease in TNF-α expression.

Figure 3

miR-221 downregulates the levels of inflammatory cytokines and oxidative stress. (a) miR-221 inhibited the expression of TNF-α and IL-6. (b) miR-221 markedly alleviated oxidative stress. ^∗∗p < 0.01 vs. sham group; ^##p < 0.01 vs. SCI+NC group.

Figure 4

TNF-α was a direct target gene of miR-221. (a) The binding sequence of TNF-α and miR-221. (b) Luciferase report assay demonstrated that miR-221 overexpression suppressed the luciferase activity of wild-type (WT) 3′-UTR of TNF-α. ^∗p < 0.05.