Pathogenic Role And Mechanism Of E2F2 In Inflammation Of Rheumatoid Arthritis Synovium

Objective1.To investigate the role and mechanisms of E2F2 in inflammation of rheumatoid arthritis.2.To investigate the role and mechanisms of E2F2 in autophagy of rheumatoid arthritis synovial fibroblasts Methods1.The expression of E2F2 and STAT1 in RASFs was inhibited by siRNA or upregulated by recombinant adenovirus vector transfection,the expression level of E2F2 and STAT1 were detected using RT-PCR and Western Blot.2.The E2F2 knockout?KO?and wild type?WT?mice were injected with collagen to induce CIA at age of 8 weeks.The degree of arthritis in mice was evaluated;HE staining and Safranin green staining were used to detect joint damage in different genotype mice;Embryos of WT or E2F2 KO mice were used for identification and primary culture in vitro to get embryonic fibroblasts?MEFs?;ELISA and RT-qPCR were used to detecte the expression of inflammatory factors IL-1?,IL-1?,and TNF-?in serum of E2F2^-/-and WT mice.3.Western Blot and RT-qPCR were performed to evaluate the effect of E2F2 on the activity of signaling pathways.ChIP-PCR and Luciferase assays were used to detect the transcriptional activity of target gene.The nuclear translocation of STAT1 and p65 were assayed by Western Blot,Co-immunoprecipitation?Co-IP?,and Immunofluorescence experiments.RT-qPCR was used to examine the effects of changes in genes or pathways on the expression of IL-1?,IL-1?and TNF-?.4.Analysis of our previous RNA-seq data revealed that E2F2 may be associated with autophagy in RASFs.Western Blot,Immunofluorescence,and transmission electron microscope were used to detect the formation of autophagy in RASFs.Western blot and RT-qPCR were performed to evaluate the effect of E2F2 on the differential genes related to autophagy,while ChIP-PCR and Luciferase assays were used to detect the transcriptional activity of target genes.Western Blot was used to detect the exchange of autophagy after high expression of E2F2 and inhibition of its tartet gene downstream..5.GST-Pull down and mass spectrometry were performed to identify E2F2 binding proteins,and then the result was confirmed using Co-IP and immunofluorescence.And effects of the binding protein on autophagy in RASFs were tested.6.RT-qPCR and Western Blot were used to confirm the effect of interacting proteins on ATG9A.ChIP-PCR and Luciferase assays were used to detect the transcriptional activity and binding domain of target gene.Transcriptional activity of ATG9A was detected by double luciferase report with siE2F2 and different concentrations of EIF5A plasmids present together.In similar manner,transcriptional activity of ATG9A by double luciferase report with si-EIF5A inhibitor and different concentrations of E2F2 plasmids presesent.E2F2 mutants of different domains was constructed to detect binding sites of E2F2 and EIF5A by Co-IP.Results1.The occurrence,severity,inflammatory cell aggregation,cartilage destruction,and the content of inflammatory factors in peripheral blood decreased in E2F2-knockout mice compared with wild type ones.The expression of IL-1?,IL-1?,and TNF-?was also suppressed in MEFs from E2F2-knockout mice.2.ChIP-PCR,double Luciferase Report,and RT-qPCR showed that E2F2 can directly bind to the promoter region of STAT1,and then regulate entry of STAT1 into nucleus,ultimately promoted the expression and secretion of IL-1?,IL-1?,and TNF-?.However,the effect of STAT1 on IL-1?,IL-1?,and TNF-?in RASFs was not so strong as STAT1,so that knock-down of STAT1 can not completely reverse the up-regulation of inflammatory factors by E2F2.Therefore,we speculated that there are other pathways besides STAT1 that mediating the inflammation.3.Analysis of RNA-seq data indicated that E2F2 can promote the activation of PI3K/AKT/NF-?B pathway.Myd88 is reported to be a key molecule mediating activation of PI3K/AKT/NF-?B pathway.As a result,ChIP-PCR and double Luciferase Report showed that E2F2 can directly bind to the promoter region of Myd88,and then regulate the activity of PI3K/AKT/NF-?B pathway,ultimately the expression and secretion of IL-1?,IL-1?,and TNF-?were promoted.We also found that E2F2 could upregulate the expression of STAT1and Myd88 through direct binding on their promoters.Moreover,E2F2 can facilitate the formation of STAT1/MyD88 complexes,and consequently activate AKT.Silencing STAT1/MyD88 or inactivating AKT could significantly attenuate the induction of IL-1?,IL-1?,and TNF-?caused by E2F2.4.RNA-seq data showed that E2F2 may be associated with autophagy.Western Blot,immunofluorescence,and transmission electron microscopy all showed that interfering with E2F2 could significantly inhibit the autophagy of RASFs.In the downstream target genes,we found that E2F2 could significantly regulate the expression of autophagy-related molecule ATG9A.ChIP-PCR and double luciferase reports shown that E2F2 could directly bind to the promoter region of ATG9A.GST-Pull down and mass spectrometry showed that EIF5A,a translation elongation factor,might be a E2F2 binding protein.,which was confirmed by Co-IP and immuno-co-localization.Western Blot,immunofluorescence and transmission electron microscopy showed that knockdown EIF5A could significantly inhibit autophagy in RASFs.And EIF5A can also regulate the autophagy through ATG9A.Western Blot,double Luciferase Report,and RT-qPCR showed that E2F2 and EIF5A can synergistically promote the transcription of ATG9A.Inhibition of EIF5A with E2F2 overexpressed resulted in significantly increased transcriptional activity of ATG9A,but inhibition of E2F2 with EIF5A overexpressed could not get the same result.Different domains of E2F2 were knocked out to find the binding domain of E2F2 to EIF5A.Conclusion1.This study further confirmed the pathological role of E2F2 in RA and identified the existence of E2F2-STAT1/MyD88-Akt axis to be closely related with the inflammatory phenotype of RASFs.2.E2F2 can promote autophagy of RASFs by directly binding to the promoter region of ATG9A.EIF5A,a translation initiation factor,can bind with E2F2 and promote autophagy by directly regulating the transcription of ATG9A.