Pathogenic Role And Mechanism Of ISG20 In Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Objective1.To Study the biological functions of the ISG20(Interferon-stimulated exonuclease gene 20)gene in the rheumatoid arthritis fibroblast-like synoviocytes(RA FLS);2.To study the regulatory mechanism of abnormal expression of ISG20 in RA FLS;3.To investigate the pathogenic mechanism of the ISG20 in regulating the activity of RA FLS.MethodsThe expression of ISG20 gene in RA FLS was inhibited by siRNA or upregulated by recombinant adenovirus vector transfection,the expression level of ISG20 was detected using RT-qPCR and Western blot.The effect of ISG20 on the proliferation in RA FLS was detected by MTS.The effect of ISG20 on CRL-1730 tube formation ability in vitro was detected by Matrigel assay.Flow cytometry was used to detect the effect of ISG20 on RA FLS cell cycly.RT-qPCR was used to detect the expression of IL-6 stimulated by ISG20,which is the key proinflammatory cytokines of RA pathogenesis.Transwell was used to detect the effect of ISG20 on cell migration and invasion.Effect of ISG20 on mRNA which related Th17 differentiation in PBMC was detected using RT-qPCR.Effect of ISG20 on osteoclasts differentiation was detected using RT-qPCR.RT-qPCR and Western blot were performed to detect the effect of the proinflammatory cytokines including IL-6,LPS and TNF-? on ISG20.To determine pathways during the stimulation process.CLI-095,PD98059,PDTC and stattic were used to block TLR4,ERK,NF-?B and STAT3 signaling pathway.RNA-sequence was performed to determine genes and pathways downstream of ISG20.Rt-qPCR method were performed to detect the expression of PTEN and PPAR? after treated with ISG20,and the expression of IL-6 regulation under these two genes.Rt-qPCR method were performed to detect the expression of miR-365 and miR-146,which is predicted by literature and bioinformatics could suppress IL-6.RT-qPCR method were performed to confirm the regulation of two miRNA by ISG20 and thier fuction to IL-6.ResultsRT-qPCR results showed that 200 ?mol/L ISG20 siRNA can significantly inhibit ISG20 expression compared to NC group(P < 0.01).ISG20 recombinant adenovirus vector(0.8 ?g/ml)can obviously increase the expression of ISG20(P < 0.01).Western blot confirmed the results at protein level.MTS assay showed that ISG20 siRNA can significantly inhibit cell proliferation of RA FLS(P < 0.05).However,ISG20 has no effect on cell proliferation when it is upregulated by recombinant adenovirus vector.Lumen formed experiments showed siISG20 can inhibit the lumen formed quantity(P < 0.01),and consistently,overexpression ISG20 have a role for promoting the lumen test results.Cells proportion in G0/G1 phase(P<0.01)increased and the S phase(P<0.01)and G2/M phase(P<0.001)cells proportion decreased after ISG20 was silenced.RT-qPCR showed that the expression of IL-6 were significantly upregulated when ISG20 was overexpressed.Moreover,we also found that siISG20 can significantly inhibit the markers related to Th17 cells differentiation including RORc,IL-17,IL-22,and IL-23(P < 0.05),and consistently,ISG20 overexpression can increase expression of these four makers and IL-21(P < 0.05).We also found that siISG20 can inhibit expression of RANK and RANKL in RA FLS,which are markers related to osteoclast differentiation(P< 0.05),and overexpression of ISG20 can increase expression of these two markers(P < 0.05).ISG20 has no effect on invasion and migration of RA FLS.IL-6,LPS,and TNF-? can stimulate the expression of ISG20(IL-6,P<0.01.LPS,P<0.01.TNF-?,P<0.05),and LPS has the most obvious effect.Inhibitors of ERK,NF-?B,and STAT3 signaling pathway could inhibit the expression of ISG20 stimulated by LPS,and ERK signaling pathway has the most obvious effect(P<0.05).TRL4 signaling pathways can also inhibit the expression of ISG20 stimulated by LPS(P<0.01).RNA-Seq results showed multiple key genes including PTEN and PPAR?,and they might be regulated by ISG20.RT-qPCR confirmed that silence of ISG20 can upregulate the expression of PTEN and PPAR?.Knockdown of these genes resulted in inhibition of IL-6.miR-365 and miR-146 were also found to be upregulated in ISG20 knockeddown RA FLS,which in turn inhibit the expression of IL-6.ConclusionISG20 in RA FLS can increase cell proliferation,secretion of inflammatory cytokine IL-6,CRL-1730 tube formation,and result in less G0/G1 arrestting.ISG20 might also play an important role in Th17 cell and osteoclast differentiation.But it has no effect on RA FLS migration and invasion.All these prompt that ISG20 might play an important role in RA.IL-6,LPS,and TNF-? can stimulate the expression of ISG20.ERK and TLR4 signaling pathway might participate the stimulation of ISG20 by LPS.RNA-Seq and bioinformatics analysis found that ISG20 may play an indirectly role in promoting the expression of IL-6,by regulation on PTEN and PPAR? gene.ISG20 may play an indirectly role in promoting the expression of IL-6,by regulation on miR-365 and miR-146.