双特异性磷酸酶8通过与促分裂原活化蛋白激酶1相互作用调控类风湿关节炎成纤维样滑膜细胞增殖 凋亡

Dual specificity phosphatases 8 directly interacts with mitogen-activated protein kinase 1 to regulate the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synovial cells

目的:探讨RA-成纤维样滑膜细胞(FLS)中双特异性磷酸酶8(DUSP8)DUSP8与促分裂原活化蛋白激酶(MAPK)1之间的相互作用,及其对RA-FLSs增殖、凋亡的影响。方法:培养RA-FLS和正常FLS,采用实时荧光定量聚合酶链式反应(RT-qPCR)、蛋白质印迹法检测两组细胞DUSP8 mRNA表达水平。采用细胞转染技术及RNA干扰技术,构建DUSP8过表达细胞系及DUSP8沉默细胞系。CCK-8法检测各组细胞增殖,流式细胞术检测各组细胞凋亡。蛋白质印迹法检测各组细胞DUSP8、MAPK1、p-MAPK1、ki-67、Bax蛋白表达水平。间接免疫荧光实验分析DUSP8与MAPK1的空间共定位,免疫共沉淀实验分析DUSP8与MAPK1蛋白之间是否存在相互作用。计量资料两组间比较采用独立样本 t检验,多组间比较采用单因素方差分析,进一步两两比较采用LSD- t检验。 结果:DUSP8在RA-FLS mRNA[(2.4±0.6)和(11.2±0.8), t=21.63, P<0.001]和蛋白表达[(0.24±0.04)和(0.74±0.08), t=9.45, P<0.001]水平均显著低于FLS。与空白对照组和过表达对照组相比,在RA-FLS细胞转染pcDNA3.1-Myc-DUSP8可显著抑制RA-FLS细胞的增殖[(90.5±5.6),(92.5±1.8),(56.4±4.4), F=138.60, P<0.001],增加细胞凋亡率[(12.7±1.4)%和(12.6±1.3)%和(27.5±3.0)%, F=16.98, P<0.001],上调细胞的DUSP8'[(0.49±0.05),(0.45±0.04)和(0.73±0.07)]、Bax[(0.39±0.06),(0.36±0.05)和(0.89±0.10)]蛋白表达水平,下调ki-67[(1.07±0.12)和(1.11±0.16)和(0.70±0.08)]、p-MAPK1/MAPK1[(0.59±0.06)和(0.65±0.07)和(0.39±0.03)]蛋白表达水平(均 P<0.001);与空白对照组和沉默对照组相比,在RA-FLS细胞转染siRNA-DUSP8可显著促进RA-FLS细胞的增殖[(90.5±5.6)和(91.1±2.9)和(128.3±4.6), F=137.50, P<0.001],降低细胞凋亡率[(12.7±1.4)%和(13.2±1.2)%和(5.4±0.7)%, F=16.98, P<0.001],下调细胞中的DUSP8[(0.492±0.048)和(0.432±0.051)和(0.102±0.024)]、Bax[(0.391±0.062)和(0.411±0.058)和(0.090±0.011)]蛋白表达水平,上调ki-67[(1.07±0.12)和(1.11±0.15)和(1.93±0.22)]、p-MAPK1/MAPK1[(0.59±0.06)和(0.68±0.06)和(0.93±0.11)]蛋白表达水平(均 P<0.05)。间接免疫荧光实验结果表明DUSP8与MAPK1均可在RA-FLS细胞质和细胞核中表达,免疫共沉淀实验验证DUSP8与MAPK1蛋白具有相互作用。 结论:DUSP8通过与MAPK1相互作用调节MAPK1磷酸化,抑制类风湿关节炎成纤维样滑膜细胞增殖,并促进凋亡。

Objective To explore the interaction between dual specificity phosphatases 8(DUSP8)and mitogen-activated protein kinase 1(MAPK1)in rheumatoid arthritis fibroblast-like synovial(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 DUSP8 mRNA and protein in the two groups of cells.DUSP8 overexpression cell lines and DUSP8 silencing cell lines were constructed using cell transfection technology and RNA interference technology,respectively.Cell counting kit 8(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 DUSP8,MAPK1,p-MAPK1,ki-67 and Bax protein in each group.The indirect immunofluorescence experiment was used to analyze the spatial co-localization of DUSP8 and MAPK1,and the co-immunoprecipitation experiment was used to analyze whether there was interaction between DUSP8 and MAPK1 protein.The t-test was used to compare the means of the two groups.One-way analysis of variance was used to compare the mean values of the three groups of samples,and then the LSD-t tests were used to compare the two groups.Results In RA-FLS,both mRNA[(2.4±0.6)vs(11.2±0.8),t=21.63,P<0.001]and protein levels[(0.24±0.04)vs(0.74±0.08),t=9.45,P<0.001]of DUSP8 were significantly lower than FLS.Compared with the blank control group and the overexpression control group,RA-FLS cells transfected with pcDNA3.1-Myc-DUSP8 could inhibit the proliferation of RA-FLS cells[(90.5±5.6)vs(92.5±1.8)vs(56.4±4.4),F=138.60,P<0.001],increase the rate of apoptosis significantly[(12.7±1.4)%vs(12.6±1.3)%vs(27.5±3.0)%,F=16.98,P<0.001],increase the expression levels of DUSP8[(0.49±0.05)vs(0.45±0.04)vs(0.73±0.07),Bax(0.39±0.06)vs(0.36±0.05)vs(0.89±0.10)]and down-regulate the expression levels of ki-67[(1.07±0.12)vs(1.11±0.16)vs(0.70±0.08),and p-MAPK1/MAPK1[(0.59±0.06)vs(0.65±0.07)vs(0.39±0.03)(all P<0.001).Compared with the blank control group and the silent control group,RA-FLS cells transfected with siRNA-DUSP8 could promote the proliferation of RA-FLS cells[(90.5±5.6)vs(91.1±2.9)vs(128.3±4.6),F=137.50,P<0.001)and decrease apoptosis rate[(12.7±1.4)vs(13.2±1.2)vs(5.4±0.7),F=16.98,P<0.001],down-regulate the expression levels of DUSP8[(0.492±0.048)vs(0.432±0.051)vs(0.102±0.024)],Bax[(0.391±0.062)vs(0.411±0.058)vs(0.090±0.011)],and up-regulate the expression levels of ki-67[(1.07±0.12)vs(1.11±0.15)vs(1.93±0.22)],p-MAPK1/MAPK1[(0.59±0.06)vs(0.68±0.06)vs(0.93±0.11)](all P<0.001).The results of indirect immunofluorescence tests showed that both DUSP8 and MAPK1 were ex-pressed in the cytoplasm and nucleus of RA-FLS.The co-immunoprecipitation study verified that DUSP8 and MAPK1 protein could interact with each other.Conclusion DUSP8 can bind to MAPK1 and regulate the abundance of active phospho-MAPK1 through its phosphatase activity and by inhibiting the proliferation of RA-FLS and promoting apoptosis.