Investigating The Role Of E2F Transcription Factor 2 Expression In Rheumatoid Arthritis

Objects: 1.To analyze the biological function of E2F2 in rheumatoid arthritis synovial fibroblasts(RASFs).2.To study the regulatory mechanism of E2F2 expression in RASFs.3.To investigate pathogenesis mechanism of E2F2 in RASFs.Methods: 1.Expressions of E2F2 in RASFs and OA SFs were detected using RT-q PCR and Western Blot;Small interference of E2F2 was transfected into RASFs to silent the expression of E2F2,and negative control si RNA was used as the negative control group(NC).After interference efficiency confirmation,MTS and Transwell were used to detect proliferation and invasion of RASFs.2.Stimulations of proinflammatory cytokines,including IL-6,TNF-?,and LPS,on the expression of E2F2 were detected using RT-q PCR and Western Blot.Inhibitors of NF-?B,STAT3,and ERK signaling pathways,(PDTC,stattic and PD98059),were used to investigate the pathway between proinflammtory cytokines and E2F2.Ch IP was used to verify the direct binding of the key signaling pathway to the E2F2 promoter region 3.After stimulated with IL-6,TNF-? and LPS,nuclear protein and cytoplasmic was obtained respectively.The distribution of E2F2 in the nucleus and cytoplasm of RASFs was then detected using Western Blot to investigate the nuclear translocation of E2F2 affected by IL-6,TNF-? and LPS.Cytokines was detected by ELISA,and the expression of downstream related genes were detected using RT-q PCR after E2F2 was silenced.The linear relationship between E2F2 and proinflammatory cytokines in RA blood samples was detected by ELISA.Ch IP was used to verify the direct binding of E2F2 to the promoter region of proinflammatory cytokines.Results: 1.The expression of E2F2 in RASFs was significantly higher than in OASFs(* P<0.05).After transfected with si RNA,E2F2 was apparently knocked-down(*P<0.05),and the interference efficiency can attain 78%.The proliferation,migration,and invasion ability of RASFs was significantly decreased(*p <0.05)after si E2F2.2.IL-6,TNF-? and LPS can stimulate the expression of E2F2 in RASFs respectively,both in m RNA and protein levels in time and concentration dependent manner(*p<0.05,**p<0.01).The induction of E2F2 in response to IL-6 and TNF-? could be significantly attenuated by PDTC,a specific inhibitor of NF-?B pathway(*p<0.05,**p<0.01).And the induction of E2F2 in response to LPS can be attenuated by PD98059,a specific inhibitor of ERK pathway.(**p<0.01).These results suggest that IL-6 and TNF-? might regulate the expression of E2F2 in RASFs both via the NF-?B pathway,while LPS might regulate the expression of E2F2 via the ERK pathway.The results of Ch IP analysis showed that p65 subunit of NF-?B could bind to the promoter of E2F2 gene directly.3.E2F2 was detected both in cytoplasm and nuclear divided protein of RASFs with and without stimulation.Interestingly,when RASFs were stimulated by LPS,IL-6 and TNF-? extracellularly,more nuclear translocation of E2F2 was observed.Notably,the tendency of E2F2 nuclear translocation was more obvious in the presence of IL-6.The expression of downstream genes,including TNF-?,IL-1?,IL-1?,IL-6,PGE2,MMP2,MMP9 and MMP13,were suppressed in E2F2 knocked-down RASFs.ELISA found that E2F2 silencing can even decrease the secretion of IL-6,IL-1?,IL-1? and TNF-? from RASFs(*p < 0.05,**p < 0.01).Induction of IL-6 by TNF-? can be suppressed by E2F2 silencing in RASFs.ELISA also tested the significant correlations between E2F2 and IL-6(r2=0.5940,P=0.0008),meanwhile between E2F2 and TNF-?(r2=0.6142,P=0.0005)in RA serum(n=15).Ch IP found that E2F2 could bind to the promoter of IL-6 gene and the binding of E2F2 to the IL-6 promoter increased significantly under TNF-? and LPS stimulation.Conclusion: In summary,in the study we demonstrated that the increased expression of E2F2 in RA synovial tissues contributes to the abnormal proliferation,invasion and cytokine production of RASFs.The induction of E2F2 in RASFs under inflamed condition further confirmed the pro-infalmmatory role of E2F2 in RA.Of note,a positive feedback loop among NF-?B/E2F2/IL-6 may exist in RASFs,finally leading to the hyper-inflammation of RA.Totally,our finding indicates E2F2 to be a potential target in the therapeutic approach of RA.Further studies focusing on E2F2 may highlight more pathological mechanisms of RA.