| Objective:SOX5 is known as a transcription factor which primarily involved in the regulation of embryonic development, cell fate, and chondrogenesis. To date,, little is known the effect of SOX5 on rheumatoid arthritis (RA). We previously reported a RANKL promoter SNP confers an elevated promoter activity after stimulation via binding to a SOX family transcription factor SOX5 and is associated with younger age at onset of RA. Subsequently, we confirmed that SOX5 could up-regulate RANKL expression in RA synovial fibroblast(SF)and participate in the bone erosion of RA. Increased SF migration and invasion plays a central role in pathogenesis of rheumatoid arthritis (RA). Here, we examine the effect of SOX5 on the migration and invasive characteristics of RA synovial fibroblasts.Methods:1. The effects of SOX5 on RASFs migration and invasion(1) Knockdown the expression level of SOX5 in RA SFs by transfecting with lentivirus-SOX5 siRNA. The effects of SOX5 on RA SFs migration and invasion were detected by Transwell corning method, wound closure motility assay and the matrigel invasion chambers method.(2) The effect of SOX5 on F-actin fibres was measured by FITC-Phalloidin staining.2. The mechanisms of SOX5 regulating RASFs migration and invasion.(1) Lentivirus-SOX5 siRNA and Adenovirus SOX5 expression vectors were used to silence and overexpress SOX5 in cell line MH7A.Then the MMP9 expressions were investigated by real-time PCR and western blot.(2) The recombinant MMP9 cytokine was added into SOX5 silenced RASFs expression to determine whether the SOX5 could promotes RA SFs migration and invasion by regulating MMP9 expression。3. The mechanisms of SOX5 modulation MMP9 expression.(1) ChIP assay was used to measure whether the SOX5 could bind to MMP9 promoter.(2) Luciferase assay was used to investigate the effect of SOX5 on MMP9 promoter activity.(3) The effect of SOX5 on MMP9 expression-related miRNA was detected by real-time PCR. The miR-15a,16 mimics was transfected into SOX5 overexpressed MH7A to determine whether the SOX5 could regulate MMP9 expression by modulation miR-15,16.4. The effect of local silencing Sox5 on Mmp9 expression and SFs invasion in CIA miceLocally silence of sox5 by injecting of lentivirus-shSOX5 into Collagen-induced arthritis (CIA) mice joint to examine the effect of SOX5 on MMP9 expression and SFs invasion in Vivo. Results:1. The effects of SOX5 on RASFs migration and invasionSilencing SOX5 could markedly decrease migration and invasion of RASFs and reduce cytoskeletal rearrangement, compared with mock transfected group.2. The mechanisms of SOX5 regulating RASFs migration and invasion.Silencing SOX5 markedly decreased the expressions of MMP2 and MMP9, especially the level of MMP9 expression.while,there was no significant differences in MMP1 and MMP3 expression. Overexpression of SOX5 significantly induced MMP-9 expression in MH7A. Migration and invasion of RASF could be markedly decreased by silencing SOX5 expression but could be reverted by added the recombinant MMP9 could revert decreased migration and invasion of RASF caused by silencing SOX5.3. The mechanisms of SOX5 modulation MMP9 expression.The results of ChIP assay and luciferase assay confirmed transcription factor SOX5 could bind the MMP9 promoter and enhance activity of MMP9 promoter. Silencing SOX5 expression in MH7A up-regulated miR-15a,16 expression and Overexpressing SOX5 expression down-regulated miR-15a,16 expression.Transfection of miR-15a mimics in MH7A significantly decreased the SOX5-induced MMP9 expression and inhibit MH7A migration. miR-16 mimics also decreased the SOX5-induced MMP9 expression,but has no significant effect on MH7A migration.4. The effect of local silencing Sox5 on Mmp9 expression and SFs invasion in CIAmiceLocal silencing SOX5 in CIA mice inhibited the SF invasion and accompanied by the markedly reduced MMP9 expression in synovium.Conclusions:1. Transcription factor SOX5 promotes RA SFs migration and invasion by regulating MMP9 expression.2. Transcription factor SOX5 could bind to MMP9 promoter and direct enhance MMP9 Transcription. On the other hand, SOX5 could indirect induce MMP9 expression by reducing miR-16 expression.3. Local suppression Sox5 expression in CIA mice inhibited the SF invasion likely by inhibiting Mmp9 expression. |
