Effect Of MiR-206 On The Migration And Invasion By Targeting TIMP3 In Rheumatoid Athritis Fibroblast-like Synoviocyte

Research background and purpose:Rheumatoid arthritis(RA)is a chronic autoimmune disease characterized by synovial hyperplasia of the joints.Pathological features include inflammatory cell infiltration,synovial tissue hyperplasia and pannus formation,leading to cartilage and bone destruction and ultimately joint dysfunction.Rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS)are the main components of synovial tissue.Under the stimulation of inflammatory factors,RA-FLS are abnormally activated and have some characteristics similar to tumor cells such as abnormal migration and invasion.They play a key role in cartilage and bone erosion.Matrix metalloproteinase tissue inhibitors(TIMPs)are natural inhibitors of matrix metalloproteinases(MMPs).Among them,TIMP3 is the unique inhibitor of almost all MMPs in the TIMPs family.Small molecule non-coding RNAs are regarded as biomarkers for autoimmune diseases,and have been reported to be involved in the regulation of cell proliferation,apoptosis,differentiation,angiogenesis and immune response.miR-206 belongs to the MyomiRs family,and accumulating evidences have demonstrated that miR-206 can regulate the migration and invasion of some tumor cells.We previously have found that the migratory and invasive ability of RA-FLS was stronger than that of normal FLS.In addition,we preformed gene chip detection combined with RT-qPCR assay to verify that miR-206 levels were significantly increased in RA-FLS when compared with those in normal FLS,while TIMP3 mRNA Levels were significantly decreased in RA-FLS when compared with those in normal FLS.Therefore,the current study aims to observe whether miR-206 can affect the migration and invasion of RA-FLS,and to clarify whether miR-206 regulates the migration and invasion of RA-FLS through targeting TIMP3.Our results might be helpful us to further understand the mechanism of cartilage and bone erosion and damage in RA patients,and might provide novel potential therapeutic strategies for RA.Methods:Human RA-FLS cell line(MH7A)and primary cultured RA-FLS obtained from RA patients were performed in following experiments.1.MH7A and primary cultured RA-FLS were incubitied with with FITC-conjured anti-human CD90 antibody and PE-conjured anti-human CD55 antibody,and verified by flow cytometry.2.The adenovirus encoding TIMP3(ad-TIMP3)were used to infect RA-FLS.The mRNA and protein levels of TIMP3 in RA-FLS were detected by RT-qPCR assay and Western Blot assay respectively.The effect of overexpression of TIMP3 on migration of RA-FLS were studied by cell scratch assay and Transwell cell migration assay,while the effect of overexpression of TIMP3 on RA-FLS invasion was investigated by Transwell cell invasion assay.3.The adenovirus encoding miR-206(ad-miR-206)were used to infect RA-FLS.The mRNA and protein levels of miR-206 in RA-FLS were detected by RT-qPCR assay and Western Blot assay,respectively.The effect of overexpression of miR-206 on migration of RA-FLS was studied by cell scratch assay and Transwell cell migration assay,and the effect of overexpression of miR-206 on RA-FLS invasion was evaluated by Transwell cell invasion assay.4.The lentiviruses encoding miR-206 inhibitor were packaged in HEK293T cell and were used to infected MH7A cells,and RT-qPCR assay was used to confirm that miR-206 expression was knocked down in MH7A cells.The effect of miR-206 knockdown on RA-FLS migration was studied by cell scratch assay and Transwell cell migration assay.The effect of miR-206 knockdown on RA-FLS invasion was investigated by Transwell cell invasion assay.5.The mRNA and protein levels of TIMP3 in RA-FLS infected with the adenovirus encoding miR-206 were detected by RT-qPCR assay and Western Blot assay.The mRNA and protein level of TIMP3 in RA-FLS infected with the lentivirus encoding miR-206 inhibitor were detected by RT-qPCR assay and Western Blot assay.6.3'UTR of TIMP3 which might be predictional target for miR-206 was amplified,and the fragment was cloned into psiCHECK-2 vector to construct psiCHECK-2-TIMP3-3'UTR-wild type(WT)recombinant vector.Moreover,psiCHECK-2-TIMP3-3'UTR-mut recombinant vector was also constructed by using the Fast Site Directed Mutagenesis Kit.After transfection with the luciferase reporter vectors psiCHECK-2-TIMP3-3 'UTR-WT or psiCHECK-2-TIMP3-3'UTR-mut in miR-206 overexpressing cells,miR-206 Knocking down cells,and normal control cells(NC),dual luciferase reporter gene assay was used to detect whether miR-206 can target TIMP3.Results:1.Data from flow cytometry showed that the percentage of MH7A expressing CD55+/CD90+ was 95.27 ± 1.34%(n=4),and the percentage of primary cultured RA-FLS expressing CD55+/CD90+ was 97.82 1.26%(n=4).Thus,both of the MH7A and primary cultured RA-FLS belongs to RA-FLS and can be used in the following studies.2.The mRNA levels of TIMP3 were significantly increased in RA-FLS infected with adenovirus encoding TIMP3.Results from the cell scratch and Transwell cell migration assays showed that overexpression of TIMP3 significantly decreased the migration of RA-FLS.Data from Transwell cell invasion assay also showed that overexpression of TIMP3 obviously reduced the invasion of RA-FLS.3.Results from RT-qPCR showed that the expression of miR-206 in RA-FLS infected with adenovirus encoding miR-206 was up-regulated.Data from cell scratch and Transwell assays showed that overexpression of miR-206 significantly promoted the migration and invasion of RA-FLS.4.Results from RT-qPCR showed that miR-206 was knocked down in the RA-FLS infected with lentivirus encoding miR-206 inhibitor.The results of cell scratch assays and Transwell assays demonstrated that knockdown of miR-206 markedly inhibited the migration and invasion of RA-FLS.5.Results from RT-qPCR and Western Blot analysis showed that the mRNAand protein levels of TIMP3 were down-regulated in miR-206 overexpressing the RA-FLS,while up-regulation of TIMP3 mRNA and protein was shown in miR-206 knocking down RA-FLS.6.DNA sequencing assay demonstrated that psiCHECK-2-TIMP3-3' UTR-WT recombinant vector and psiCHECK-2-TIMP3-3'UTR-mut recombinant vector were successfully constructed.Data from dual luciferases reporter gene assay in which the Renilla luciferase activity was measured and normalized to firefly luciferase activity,showed that the relative Renilla luciferase activities were significantly reduced in psiCHECK-2-TIMP3-3'UTR-WT-transfected miR-206 overexpressing cells,while no significant alteration was shown in the psiCHECK-2-TIMP3-3'UTR-mut-transfected cells in comparison with those in NC cells.The relative Renilla luciferase activities were increased in psiCHECK-2-TIMP3-3'UTR-WT transfected miR-206knocking down cells,but not in the psiCHECK-2-TIMP3-3'UTR-mut transfected cells in comparison with those in NC cells.Considering that miR-206 overexpression down-regulated the expression of TIMP3 as mentioned above,our results strongly indicated that TIMP3 might be a direct target of miR-206 in RA-FLS cells,and the inhibition of miR-206 to TIMP3 expression requires miR-206 directly binding to the predicted binding sites on the TIMP33'UTR.Conclusions:1.miR-206 can affect the migration and invasion of RA-FLS.2.miR-206 regulates the migration and invasion of RA-FLS through targeting TIMP3.