C-Fos directly interacts with mitogen activated protein kinase 14 to regulate the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synovial cells / 中华风湿病学杂志

ABSTRACT

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 Mean ± 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.