C-Fos与丝裂原活化蛋白激酶14相互作用对类风湿关节炎成纤维样滑膜细胞增殖凋亡的影响

C-Fos directly interacts with mitogen activated protein kinase 14 to regulate the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synovial cells

目的探讨C-Fos与丝裂原活化蛋白激酶14(MAPK14)相互作用参与调控类风湿关节炎成纤维样滑膜细胞(RA-FLS)增殖、凋亡的机制。方法采用实时荧光定量聚合酶链式反应PCR(RT-qPCR)、蛋白质印迹法检测RA-FLS和正常成纤维样滑膜细胞(FLS)细胞C-Fos mRNA和蛋白表达水平。将RA-FLS细胞分为C-Fos过表达组(转染pcDNA3.1-Myc-C-Fos质粒)、过表达对照组(转染pcDNA3.1-Myc空质粒)、C-Fos沉默组(转染siRNA-C-Fos)、沉默对照组(转染siRNA-NC)和空白对照组(未加任何处理)。细胞计数试剂(CCK-8)检测各组细胞增殖能力,流式细胞实验检测各组细胞凋亡的变化,蛋白质印迹法检测各组细胞C-Fos、MAPK14、p-MAPK14、ki-67、Bax蛋白表达水平。间接免疫荧光实验检测C-Fos和MAPK14在细胞中的定位关系。免疫共沉淀方法检测C-Fos-MAPK14的相互作用关系。采用GraphPad Prism 5统计学软件进行数据分析,正态分布的数据以x(-)±s表示,2组间比较采用独立样本t检验;多组比较采用应采用方差分析,进一步两两比较采用LSD-t检验。结果RA-FLS细胞中C-Fos mRNA(5.37±0.91)相对表达量明显高于FLS细胞(1.46±0.32)(t=9.94,P<0.001)。C-Fos在RA-FLS蛋白水平(1.12±0.15)均显著高于FLS(0.81±0.07)(t=3.18,P=0.017)。与空白对照组和过表达对照组相比,过表达C-Fos可显著促进RA-FLS细胞增殖,抑制细胞凋亡率,上调细胞p-MAPK14、ki-67蛋白水平,下调细胞Bax蛋白水平(均P<0.05);与空白对照组和沉默对照组相比,沉默C-Fos可显著抑制RA-FLS细胞的增殖,增加细胞凋亡率,下调细胞p-MAPK14、ki-67蛋白水平,上调细胞Bax蛋白表达水平(均P<0.05)。间接免疫荧光实验结果表明C-Fos与MAPK14均可在RA-FLS细胞核中表达,免疫共沉淀实验验证C-Fos与MAPK14蛋白具有相互作用。结论C-Fos与MAPK14相互作用促进磷酸化MAPK14蛋白表达,从而促进RA-FLS增殖,并抑制凋亡。

Objective To explore the interaction between C-Fos and mitogen activated protein kinase 14(MAPK14)in rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS),and its effect on the proliferation and apoptosis of RA-FLSs.Methods RA-FLS and normal fibroblast-like synovial cells(FLS)were cultured.Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)and Western blotting were used to detect the expression levels of C-Fos mRNA and protein in the two groups.RA-FLS cells were divided into C-Fos overexpression group(transfected with pcDNA3.1-Myc-C-Fos plasmid),overexpression control group(transfected with pcDNA3.1-Myc empty plasmid),and C-Fos silent group(transfection siRNA-C-Fos),silence control group(transfection siRNA-NC)and blank control group(without any treatment).CCK-8 method was used to detect cell proliferation in each group,and flow cytometry was used to detect cell apoptosis in each group.Western blotting was used to detect the expression levels of C-Fos,MAPK14,p-MAPK14,ki-67 and Bax protein in each group.The indirect immunofluorescence experiment analyzed the spatial co-localization of C-Fos and MAPK14,and the co-immunoprecipitation experiment analyzed whether there was interactions between C-Fos and MAPK14 protein.The results of the experimental data were analyzed by Graph Pad Prism 5.0 software.The data of normal distribution was shown as Meanx(-)±standard deviation,and the comparison between the two independent samples using the t test.One-way Analysis of Variance(ANOVA)was used for overall comparison among the multiple groups in the experimental group,and LSD-t test was used for pair comparison within the group.P<0.05 indicated that the difference was statistically significant.Results The mRNA levels of C-Fos(5.37±0.91)in RA-FLS were significantly higher than FLS(1.46±0.32)(t=9.94,P<0.001).The protein levels of C-Fos(1.12±0.15)were significantly higher than FLS(0.81±0.07)(t=3.18,P=0.017).Compared with the blank control group and the overexpression control group,RA-FLS cells transfected with pcDNA3.1-Myc-C-Fos could promote the proliferation of RA-FLS cells,inhibit apoptosis,significantly up-regulate the expression levels of C-Fos,p-MAPK14,ki-67,and significantly down-regulate cellular Bax protein levels(all P<0.05).Compared with the blank control group and the silent control group,RA-FLS cells transfected with siRNA-C-Fos could inhibit the proliferation of RA-FLS cells,promote apoptosis,down-regulate the expression levels of C-Fos,p-MAPK1,ki-67,and up-regulate the cellular Bax protein expression level(all P<0.05).The results of indirect immunofluorescence experiments showed that both C-Fos and MAPK14 could be expressed in the nucleus of RA-FLS.The co-immunoprecipitation experiment verified that C-Fos and MAPK14 protein interact with each other.Conclusion The interaction of C-Fos-MAPK14 promotes the autophosphorylation of MAPK14,thereby promoting the proliferation of rheumatoid arthritis fibroblast-like synovial cells and inhibiting apoptosis.