EFFECT OF miR-663b ON INTERLEUKIN-1β-INDUCED INFLAMMATORY RESPONSE AND APOPTOSIS OF NUCLEUS PULPOSUS CELLS AND ITS MECHANISM

Abstract

Objective To investigate the effect of miR-663b on the inflammatory response and apoptosis of nucleus pulposus cells (NPCs) induced by interleukin-1β (IL-1β) and its mechanism. Methods According to the different treatment me-thods, NPCs were divided into group A (no treatment), group B (induced by IL-1β), group C (IL-1β induction+miR-663b mimic transfection), and group D (IL-1β induction+miR-633b NC transfection). RT-qPCR was used to measure the expression levels of miR-663b, inflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β], type Ⅱ collagen, and po-lysaccharide in NPCs; CCK-8 assay and TUNEL staining were used to observe the proliferation and apoptosis of NPCs; Western blotting was used to measure the protein expression level of interleukin-1 receptor 1 (IL1R1) in NPCs. TargetScan database was used to predict the potential binding sites between miR-663b and IL1R1. The 293T cells were divided into group E (transfected with IL1R1-wt plasmid+miR-663b mimic), group F (transfected with IL1R1-wt plasmid+miR-663b mimic), group G (transfected with IL1R1-mut plasmid+miR-663b mimic), and group H (transfected with IL1R1-mut plasmid+mimic NC), and the luciferase activity of NPCs was measured for each group. Results RT-qPCR results showed that compared with groups A, B, and D, group C had a significant increase in the relative expression level of miR-663b (t=9.41-22.93,P<0.01), and compared with groups B and D, group C had significant changes in the relative expression levels of TNF-α, IL-6, IL-1β, type Ⅱ collagen, and polysaccharide (t=3.17-32.51,P<0.01). CCK-8 assay and TUNEL staining showed that compared with groups B and D, group C had a significant increase in the proliferation of NPCs (t=3.14,3.96,P<0.01) and a significant reduction in the apoptosis of NPCs (t=4.28,168.61,P<0.01). RT-qPCR and Western blotting showed that compared with groups B and D, group C had signi-ficant reductions in the relative protein expression levels of IL1R1 and IL1R1 in NPCs (t=6.39-12.84,P<0.01). Dual-luciferase reporter assay showed that group E had a significantly lower luciferase activity than groups F, G, and H (t=10.62-16.27,P<0.01). Conclusion This study shows that miR-663b may downregulate the expression of IL1R1 in NPCs through targeted bin-ding to IL1R1 and slow down the inflammatory response and apoptosis of NPCs.

Keywords

• micrornas|nucleus pulposus|gene expression regulation|cell proliferation|apoptosis|receptors, interleukin-1|intervertebral disc degeneration