miR-214-5p靶向SIRT2介导白细胞介素-1β诱导软骨细胞损伤的机制研究

Mechanism of miR-214-5p targeting SIRT2-mediated interleukin-1β-induced chondrocyte injury

目的探讨微小RNA-214-5p(miR-214-5p)对白细胞介素-1β(IL-1β)诱导的软骨细胞损伤的影响及其分子机制。方法分离培养SD大鼠关节软骨细胞,用IL-1β处理细胞,采用qRT-PCR与Western blot检测miR-214-5p与SIRT2表达。软骨细胞分别转染anti-miR-214-5p、pcDNA-SIRT2,检测IL-1β诱导后细胞中miR-214-5p与SIRT2表达,采用流式细胞术检测细胞凋亡。利用生物信息学预测miR-214-5p的靶基因,应用双荧光素酶报告实验及Western blot方法对靶基因进行验证。将si-SIRT2转染软骨细胞,检测IL-1β诱导后软骨细胞凋亡率。采用酶联免疫吸附法(ELISA)检测细胞中IL-6、TNF-α、IFN-γ的含量;采用Western blot检测细胞中Bax、Bcl-2表达。结果IL-1β处理软骨细胞后,细胞中miR-214-5p表达水平升高,SIRT2表达水平降低;IL-1β处理后的软骨细胞凋亡增多,IL-6、TNF-α、IFN-γ含量与Bax蛋白水平升高,Bcl-2蛋白水平降低;抑制miR-214-5p表达或SIRT2过表达的软骨细胞经IL-1β诱导后,细胞凋亡率降低,IL-6、TNF-α、IFN-γ含量与Bax蛋白水平降低,Bcl-2蛋白水平升高;miR-214-5p过表达可抑制含miR-214-5p结合位点荧光报告载体的强度,将结合位点突变后抑制作用消失;抑制SIRT2表达可逆转抑制miR-214-5p表达对IL-1β诱导软骨细胞损伤的保护作用。结论抑制miR-214-5p表达可靶向SIRT2对IL-1β诱导的软骨细胞损伤发挥保护作用,其作用机制与减少软骨细胞凋亡及抑制炎性反应有关。

Objective To investigate the effect of microRNA-214-5p(miR-214-5p)on interleukin-1β(IL-1β)-induced chondrocyte injury and its molecular mechanism.Methods The articular chondrocytes of SD rats were isolated and cultured,and the cells were treated with IL-1β.The expression of miR-214-5p and SIRT2 was detected by qRT-PCR and Western blot.The chondrocytes were transfected with anti-miR-214-5p and pcDNA-SIRT2,respectively.The expression of miR-214-5p and SIRT2 in IL-1β-induced cells was detected,and apoptosis was detected by flow cytometry.The target gene of miR-214-5p was predicted by bioinformatics,and the target gene was verified by dual luciferase reporter assay and Western blot.The si-SIRT2 was transfected into chondrocytes,and the apoptosis rate of chondrocytes induced by IL-1βwas measured.The levels of IL-6,TNF-αand IFN-γin cells were detected by enzyme-linked immunosorbent assay(ELISA).The expression of Bax and Bcl-2 in the cells was detected by Western blot.Results After treatment of chondrocytes with IL-1β,the expression level of miR-214-5p was increased and the expression level of SIRT2 was decreased.The apoptosis of chondrocytes increased after IL-1βtreatment,and the contents of IL-6,TNF-αand IFN-γwere increased.The expression level of Bax is increased and the expression level of Bcl-2 was decreased.When chondrocytes inhibiting miR-214-5p expression or SIRT2 overexpression were induced by IL-1β,the apoptosis rate decreased,and the levels of IL-6,TNF-α,IFN-γand Bax decreased,but the expression level of Bcl-2 was elevated.Overexpression of miR-214-5p inhibited the intensity of the fluorescent reporter vector containing the miR-214-5p binding site,and the inhibition disappears after the binding site was mutated.Inhibition of SIRT2 expression reversibly inhibited the protective effect of miR-214-5p expression on IL-1β-induced chondrocyte injury.Conclusion Inhibition of miR-214-5p expression can target SIRT2 to protect IL-1β-induced chondrocyte injury,and its mechanism of action is related to reducing chondrocyte apoptosis and inhibiting inflammatory response.