| Objective: Inflammation is an important pathological mechanism of intervertebral disc degeneration,and overexpression of pro-inflammatory cytokines can accelerate apoptosis of nucleus pulposus cells.This study focused on the role and pathological mechanism of miRNA(mi R)-663 b in interleukin-1(IL-1)-induced inflammatory response and the apoptosis of nucleus pulpocytes.Methods: The gradient of IL-1 concentration and treatment time was set,the expression of miR-663 b was detected by RT-qPCR,and the optimal modeling concentration and induction time were selected.Then,according to different treatments of nucleus pulposus cells,they were divided into 6 groups: A,B,C,D,E and F.Group A was normal group without any treatment.Group B: control group treated with IL-1 only;Group C: IL-1+mi R663 b mimic group;Group D: IL-1+mi R663 b mimic negative control group(mimic NC);Group E:IL-1+mi R663 b inhibitor(mi R663 b inhibitor)group;Group F: IL-1+mi R663 b inhibitor negative control(inhibitor NC)group.The transfection efficiency of miRNA-663 b and the expression of inflammatory cytokines TNF-α,IL-6,IL-1β,and cell matrix type II collagen and polysaccharide protein genes were detected by RT-PCR.CCK8 and TUNEL staining were used to detect cell proliferation and apoptosis in each group.Western-bolt was used to detect the expression of IL1R1 protein and NF-κB pathway related protein.Target Scan database was used to predict the potential binding sites between miR-663 b and IL1R1,and the plasmid containing IL1R1 wild-type gene(IL1R1-WT)and IL1R1 mutant gene(IL1R1-mut)was constructed.293 T cells were transfected with IL1R1-wt+miR-663 b mimic,IL1R1-wt+mimic NC,IL1R1-mut+ miR-663 b mimic,and IL1R1-mut+ mimic,respectively NC,the luciferase activity of each group was detected to analyze the targeted regulatory effect of miR-663 b on IL1R1.Results: With the increase of IL-1 concentration and treatment time,the relative expression of miR-663 b decreased in an induction dose-and time-dependent manner.In the concentration gradient experiment,the difference between the groups was statistically significant(F=108.2,P<0.01),but when the IL-1 concentration was more than 10ng/ml,there was no significant difference between the two groups(P>0.05).In the time gradient experiment,the difference between the groups was statistically significant(F=24.33,P< 0.01),but there was no significant difference when the treatment time was more than 24h(P> 0.05).Therefore,we chose to treat nucleus pulposus cells with 10ng/ml IL-1 for 24 h to induce inflammation of nucleus pulposus cells.Among the six different treatment groups A,B,C,D,E,and F,RT-qPCR results showed that the expression of miR-663 b in group C was significantly increased(P<0.01),but significantly decreased in group E(p<0.05),indicating that the transfection effect was good.However,the expression of inflammatory factors in group C with overexpression of miR-663 b was significantly inhibited compared with other groups(p<0.05).In group E with miR-663 b inhibitor,the expression of inflammatory factors was significantly higher than other groups(p<0.01).The expression of type 2 collagen and polysaccharide protein in group C was significantly higher than that in other groups(p<0.01).The contents of type II collagen and polysaccharide in group E were significantly lower than those in other groups(p<0.01).CCK-8 cell proliferation assay showed that the cell viability of group C was significantly higher than that of the other groups(p<0.01),while the cell viability of group E was significantly inhibited compared with other groups(p<0.01).TUNEL staining results showed that the number of TUNEL staining positive cells in group C was significantly lower than that in other groups(p<0.01);The number of TUNEL positive cells in group E was significantly higher than that in other groups(p<0.01).Dual luciferase reporter gene assay showed that the luciferase activity of IL1R1-wt+miR-663 b mimic group was significantly lower than that of other groups(F=71.07,P<0.01).RT-qPCR results showed that the expression of IL1R1 in group C was decreased(p<0.01),and the expression of IL1R1 in group E was significantly up-regulated(p<0.01).Western blot analysis showed that the relative expression of IL1R1 protein in group C was significantly decreased(p<0.01),and the relative protein expression ratios of P-p65/p65 and P-IκBα/IκBαin group C were significantly decreased(P<0.05).The relative expression of IL1R1 protein in group E was increased(p<0.01).The relative protein expression ratios of P-p65/p65 and P-IκBα/IκBα were also significantly increased(P<0.05).Conclusions: MicroR-663 b down-regulates the expression of IL1R1 through targeted binding to IL1R1 and reduces the phosphorylation level of the nuclear factor kappa B pathway-related protein kinases,thereby reducing apoptosis and the inflammatory response of nucleus pulpocytes.Therefore,miR-663 b may be a promising therapeutic target for the treatment of intervertebral disc degeneration. |
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