miR-149-3p调控JNK/p38 MAPK信号通路对髓核细胞凋亡、自噬和炎性反应的影响

The effect of miR-149-3p on the apoptosis, autophagy and inflammation of nucleus pulposus cells by regulating the JNK/p38 MAPK signaling pathway

目的探讨miR-149-3p通过调控JNK/p38 MAPK信号通路对髓核细胞凋亡、自噬和炎症反应的影响。方法体外分离大鼠髓核细胞,用IL-1β浓度为10 ng/ml建立模型组,细胞分为Con组(空白对照)、IL-1β组(IL-1β处理)、IL-1β+miR-NC组(IL-1β处理,转染miR-NC)、IL-1β+miR-149-3p组(IL-1β处理,转染miR-149-3p)、IL-1β+miR-149-3p+Anisomycin组(IL-1β和通路激活剂Anisomycin处理,转染miR-149-3p)。采用qRT-PCR检测miR-149-3p相对表达水平;MTT、流式细胞术实验检测细胞增殖和凋亡;Western blot检测Bax、Bcl-2、Beclin 1、LC3II/LC3I、JNK/p38 MAPK信号通路相关蛋白表达;ELISA检测TNF-α、IL-6、IL-8表达水平。结果IL-1β组内miR-149-3p相对表达量、细胞活性、Bcl-2蛋白表达明显低于Con组,凋亡率、Bax、Beclin 1、LC3II/LC3I蛋白表达、TNF-α、IL-6、IL-8表达水平、p-JNK、p-p38 MAPK蛋白表达明显高于Con组(P<0.05)。IL-1β+miR-149-3p组内miR-149-3p相对表达量、细胞活性、Bcl-2蛋白表达明显高于IL-1β+miR-NC组(P<0.05),凋亡率、Bax、Beclin 1、LC3II/LC3I蛋白表达、TNF-α、IL-6、IL-8表达水平、p-JNK、p-p38 MAPK蛋白表达明显低于IL-1β+miR-NC组。IL-1β+miR-149-3p+Anisomycin组的p-JNK、p-p38 MAPK蛋白表达、凋亡率、Bax、Beclin 1、LC3II/LC3I蛋白表达、TNF-α、IL-6、IL-8表达水平明显高于IL-1β+miR-149-3p组,细胞活性、Bcl-2蛋白表达明显低于IL-1β+miR-149-3p组(P<0.05)。结论上调miR-149-3p能促进IL-1β诱导的髓核细胞增殖,抑制细胞凋亡、自噬和炎性反应,其作用机制可能与激活JNK/p38 MAPK信号通路有关。

Objective To investigate the effect of miR-149-3p on the apoptosis,autophagy and inflammation of nucleus pulposus cells by regulating the JNK/p38 MAPK signaling pathway.Methods Primary rat nucleus pulposus cells were isolated and induced with 10ng/ml IL-1β.Cells were divided into Con group(blank control),IL-1βgroup(IL-1βinduction),IL-1β+miR-NC group(IL-1βinduction+transfection with miR-NC),IL-1β+miR-149-3p group(IL-1βinduction+transfection with miR-149-3p),and IL-1β+miR-149-3p+Anisomycin group(induction with IL-1βand the JNK/p38 activator Anisomycin+transfection with miR-149-3p).Quantitative reverse transcription polymerase chain reaction(qRT-PCR)was performed to detect mRNA level of miR-149-3p.MTT assay and flow cytometry were conducted to detect cell proliferation and apoptosis,respectively.Western blot was used to detect protein expressions of Bax,Bcl-2,Beclin 1,LC3II/LC3I,and key proteins in the JNK/p38 MAPK signaling pathway.The enzyme-linked immunosorbent assay(ELISA)was applied to detect positive expressions of TNF-α,IL-6 and IL-8.Results The relative expression of miR-149-3p,cell activity,and the protein expression of Bcl-2 in IL-1βgroup were significantly lower than those in Con group.The apoptosis rate,protein expressions of Bax,Beclin 1,LC3II/LC3I,p-JNK,and p-p38,and positive expressions of TNF-α,IL-6 and IL-8 were significantly higher in IL-1βgroup than those of Con group.The relative expression of miR-149-3p,cell activity,and the protein expression of Bcl-2 in IL-1β+miR-149-3p group were significantly higher than those of IL-1β+miR-NC group.The apoptosis rate,protein expressions of Bax,Beclin 1,LC3II/LC3I,p-JNK,and p-p38,and positive expressions of TNF-α,IL-6 and IL-8 were significantly lower in IL-1β+miR-149-3p group than those of IL-1β+miR-NC group.Protein expressions of p-JNK,p-p38,Bax,Beclin 1 and LC3II/LC3I,apoptosis rate,and positive expressions of TNF-α,IL-6 and IL-8 were significantly higher in IL-1β+miR-149-3p+Anisomycin group than those of IL-1β+miR-149-3p group,while cell activity and protein expression of Bcl-2 were significantly lower.Conclusion Up-regulation of miR-149-3p can promote the proliferation of IL-1β-induced nucleus pulposus cells,inhibit cell apoptosis,autophagy and inflammatory response by activating the JNK/p38 MAPK signaling pathway.