Function And Mechanism Of Circ0088194 In Promoting Migration And Invasion Of Fibroblast-like Synoviocytes In Rheumatoid Arthritis

Rheumatoid arthritis（RA）is a chronic and systemic autoimmune disease characterized by synovial membrane inflammation and destruction of joint bone and cartilage.It can cause joint stiffness,deformity,and loss of function and also cause damage to multiple organs and systems such as the heart,lungs,and kidneys,seriously endangering the health and quality of life of patients.The global prevalence of RA is 0.24%.At present,its pathogenesis is not yet clear.The pathogenesis of RA originates from synovial tissue adjacent to the joint and then spreads to cartilage and bone.Fibroblast-like synoviocytes（FLS）in the synovial membrane lining play a vital role in the pathogenesis of RA.In the inflammatory process of RA,activated FLS exhibits biological behavior similar to tumor cells,and its migration and invasion ability are significantly enhanced.RA-FLS migrates to the surface of bone and cartilage under the action of a variety of inflammatory mediators and cytokines and produces proteolytic enzymes,thereby causing erosion of cartilage and bone,leading to joint destruction.Activated RA-FLS can also migrate to distant joints and has a particular impact on the destruction of multiple joints.Therefore,it is necessary to clarify the possible molecular mechanism of RA-FLS migration and invasion,and it may identify new target molecules for the early treatment of rheumatoid arthritis.Circular RNA（circular RNA,circRNA）is a class of closed circular non-coding RNA molecules that does not have a 5’end cap and a 3’polyA tail and is formed by a covalent bond.CircRNA is widely present in eukaryotic cells,with stable structure,tissue specificity,disease specificity,and timing specificity.CircRNA can participate in regulating the development and progression of many diseases.They can act as miRNA sponges,protein binding,and regulation of transcription.A small number of circular RNAs can also participate in gene expression by encoding proteins.Three different samples of RA-FLSs and OA-FLSs were used for microarray analysis.We then verified the microarray results,and Circ₀088194 was chosen for further investigation.Subsequently,the effect of Circ₀088194 on the migration and invasion of RA-FLS was studied,and the "miRNA" sponge mechanism was used as an entry point to explore the role of Circ₀088194 in the development and progression of RA and to find potential therapeutic targets for the treatment.This topic is divided into the following four parts:Part 1:Expression profile of circular RNA in fibroblast-like synoviocytes of RA Objective:Differentially expressed circRNA was detected in human rheumatoid arthritis fibroblast-like synoviocytes（RA-FLS）and osteoarthritis fibroblast-like synoviocytes（OA-FLS）.Methods:Three RA synovial tissues and three OA synovial tissues matched for age and sex were selected.RA-FLS and OA-FLS were isolated and cultured,respectively.CircRNA microarray was performed to detect the differentially expressed circRNA in RA-FLS and OA-FLS.Using real-time fluorescent quantitative PCR（qRT-PCR）to verify the microarray results by designing divergent primers spans the back-splice junction of circRNAs.Sanger sequencing is used to confirm whether the circRNA confirms the splice junction.Spearman rank correlation was used to analyze the association of Circ₀088194 expression with DAS28 score in RA patients.Finally,the subcellμlar localization of circRNA was performed by RNA fluorescence in situ hybridization.Results:1.Seven circRNAs were identified as being differentially expressed（fold change>2.5 and p<0.05）in circRNA microarray between RA-FLSs and OA-FLSs,of which 85.7%are derived from exons.2.The results showed that Circ₀088194 and Circ₀088200 were elevated in RA-FLSs compared with those in OA-FLSs,and the expression level of Circ₀088194 was more significantly increased.3.Sanger sequencing confirmed that the sequence contained a segment of the adaptor sequence of tcactgccaagttcacaacagaagccgaaccggaagttgac.4.The Circ₀088194 levels correlated positively with the disease activity score in 28 joints（DAS28）（r=0.683,p=0.043）.5.Circ₀088194 is highly expressed in the cytoplasm of RA-FLSs by RNA fluorescence in situ hybridization.Conclusion:Compared with OA-FLS,the expression of circ 0088194 in RA-FLS was increased,and circ₀088194 is associated with RA disease severity,suggesting that circ₀088194 may play an important role in the development of RA disease.Part 2:Effect of Circ 0088194 on migration and invasion of RA-FLSObjective:To investigate the effect of Circ₀088194 on the migration and invasion of RA-FLS.Methods:1.An adenoviral vector encoding Circ₀088194 was constructed and transfected into RA-FLS.The expression level of Circ₀088194 was detected by qRT-PCR to ensure the effectiveness of the overexpression vector.2.After overexpression of Circ₀088194,Transwell migration and Matrigel invasion assays were used to evaluate the migratory and invasive activity of RA-FLSs.3.Three siRNAs targeting the junction sites of Circ 0088194 were designed and transfected into RA-FLS,then the expression level of Circ0088194 was verified by qRT-PCR to ensure interference efficiency.4.One siRNA with the highest and stable interference efficiency was selected and transfected into RA-FLS.The migration and invasion activities of RA-FLS were observed by Transwell migration and Matrigel invasion assays.Results:1.Compared with the empty vector,Circ₀088194 expression in RA-FLSs increased significantly after transduction with an adenoviral vector expressing Circ₀088194.2.Compared with the empty vector,overexpression of Circ₀088194 significantly promoted the migration and invasion of RA-FLSs.3.Compared with the negative control,all three siRNAs could knock down the expression level of Circ0088194 to varying degrees.Si Circ0088194#1 had the most significant interference effect.4.Compared with the negative control,knockdown of Circ₀088194 significantly inhibited RA-FLS migration and invasion.Conclusion:Overexpression of Circ₀088194 promotes the migration and invasion of RA-FLS significantly.Part 3:The mechanism of RA-FLSs migration and invasion regulated by Circ0088194.3.1 Circ₀088194 promotes the migration and invasion of RA-FLSs by regulating the expression of MMP2Objective:To investigate the downstream key gene regulated by Circ₀088194.Method:1.We performed a protein array analysis in three overexpressing Circ₀088194 RA-FLSs compared with three control RA-FLSs to detect the differentially expressed proteins.2.qRT-PCR was used to analyze the mRNA expression of proteins with significant expression differences in protein array.3.The MMP2 mRNA and protein expression levels were examined in RA-FLSs transfected with overexpression or knockdown of Circ₀088194.4.RA-FLSs were co-transfected with the adenovirus expressing Circ₀088194 or MMP2 siRNA.The expression levels of MMP2 and the migratory and invasive activity of RA-FLSs were detected by qRT-PCR,western blotting,transwell migration,and Matrigel invasion assay,respectively.Results:1.The protein array analysis revealed that thirteen proteins were significantly differentially expressed,of which nine were downregulated,while four were upregulated.2.The qRT-PCR results showed that after Circ₀088194 overexpression,only MMP2 levels increased in RA-FLSs,and there was no significant difference in the mRNA expression levels of the other two proteins（TNF-α converting enzyme（TACE）,platelet-derived growth factor AA PDGF-AA）.3.Compared with the empty vector,MMP2 mRNA and protein levels were increased after overexpression of Circ0088194,and western blotting and qRT-PCR showed that knockdown of Circ₀088194decreased the expression of MMP2.4.Co-transfected with MMP2 siRNA decreased the mRNA and protein expression levels of MMP2 induced by overexpression of Circ₀088194 and reversed Circ₀088194 overexpression-induced migration and invasion.Conclusion:Circ₀088194 promotes the migration and invasion of RA-FLSs by regulating the expression of MMP2.3.2 Circ₀088194 sponges miR-766-3p in RA-FLSsObjective:To investigate the miRNA regulated by Circ0088194.Method:1.Using three publicly available databases（TargetScan,Miranda,and circular RNA interactome）and bioinformatics analysis to predict miRNAs that might bind to Circ₀088194 and the 3’-untranslated region（UTR）of MMP2 mRNA.2.qRT-PCR determination of the expression of miRNAs in RA-FLSs transfected with overexpression or knockdown of Circ₀088194.3.According to the predicted binding sites of miRNAs to Circ0088194 on the bioinformatics website,wild-type（WT）or mutant（MUT）miRNA target sequences of Circ₀088194 were constructed and cloned into luciferase reporter.Then we performed dual-luciferase assays to confirm the binding between miRNA and Circ₀088194.4.The direct interaction between the miRNA and Circ0088194 was validated by qRT-PCR after the Ago2 RNA immunoprecipitation（RIP）experiment.5.RA-FLSs were transfected with the adenovirus encoding Circ₀088194 or Circ₀088194 with mutated miRNA binding sites.The migratory and invasive activities of RA-FLSs were evaluated by Transwell migration and Matrigel invasion assays.Results:1.Two miRNAs that might bind to Circ₀088194 and the 3’-UTR of MMP2 mRNA were predicted by bioinformatics,and miR-766-3p was chosen for further investigation.2.Compared with the empty vector,miR-766-3p expression decreased when Circ₀088194 was overexpressed,while miR-766-3p expression increased in RA-FLSs after Circ₀088194 knockdown.3.A dual-luciferase assays reporter was used to confirm the binding between Circ₀088194 and miR-766-3p.Co-transfected with Circ₀088194-WT and miR-766-3p mimics significantly decreased the luciferase activity of RA-FLS.4.Anti-AGO2 antibodies can precipitate AGO2 protein from cell lysates,Circ₀088194 and miR-766-3p were enriched in the AGO2 immunoprecipitate.5.The migratory and invasive activities of RA-FLSs were significantly decreased after overexpression of Circ₀088194 with mutated miR-766-3p binding sites,with no significant difference compared with the migration and invasion ability of the empty vector.Conclusion:Circ₀088194 acts as a sponge for miR-766-3p.3.3 MiR-766-3p inhibits the migration and invasion of RA-FLSs by targeting MMP2.Objective:To verify that miR-766-3p targeted regulation of MMP2 inhibited migration and invasion of RA-FLS.Method:1.Transfecting RA-FLSs constructed overexpressed and knockdown miRNA with miRNA mimics or inhibitors,respectively.Using qRT-PCR and Western blotting assays to determine MMP2 expression in RA-FLSs.2.We performed dual-luciferase assays to confirm the binding between miRNA and 3’-UTR of the MMP2 mRNA.3.Assays of RA-FLSs migration and invasion in cells transfected with miRNA mimics or inhibitors,respectively.Results:1.Transfection of miR-766-3p mimics reduced the expression of MMP2 mRNA and protein in RA-FLSs.Conversely,transfection of miR-766-3p inhibitors increased the expression of MMP2 mRNA and protein.2.A dual-luciferase assays reporter was used to confirm the binding between miR-766-3p and the 3’-UTR of MMP2 mRNA.3.Transwell assays revealed that miR-766-3p reduced RA-FLS migration and invasion,while the miR-766-3p inhibitors increased RA-FLS migration and invasion.Conclusion:miR-766-3p inhibits the migration and invasion of RA-FLSs by targeting MMP2.3.4 Circ₀088194 promotes RA-FLSs migration and invasion via the miR-766-3p/MMP2 AxisObjective:To test whether Circ0088194 promotes RA-FLS migration and invasion via the miR-766-3p/MMP2 axis.Method:1.RA-FLSs were co-transfected with the adenovirus expressing Circ 0088194 or MMP2 siRNA,then the expression levels of MMP2 was detected by qRT-PCR and western blotting.2.RA-FLSs were co-transfected with the adenovirus expressing Circ₀088194 or MMP2 siRNA,then the migratory and invasive activity of RA-FLSs were evaluated by transwell migration and Matrigel invasion assay.Results:1.MiR-766-3p mimic co-transfection blocked the upregulation of the MMP2 mRNA and protein levels induced by Circ₀088194 overexpression in RA-FLSs.2.Transfection of Circ₀088194 promoted RA-FLSs migration and invasion,which could be blocked by co-transfection with miR-766-3p mimics.Conclusion:Circ₀088194 promotes RA-FLSs migration and invasion via the miR-766-3p/MMP2 Axis.Part 4:N6-methyladenosine modification of Circ0088194 in RA-FLSObjective:To detect the level of N6-methyladenosine（m6A）modification of Circ0088194 in RA-FLS and its effect on the stability of Circ0088194Methods:The expression levels of the four most essential enzymes in m6A modification（METL3,METTL14,FTO,and ALKBH5）in RA-FLS and OA-FLS were detected by qRT-PCR.2.To test the level of m6A modification in RA-FLS and OA-FLS by methylated RNA immunoprecipitation,followed by qRT-PCR of Circ₀088194.Results:1.The qRT-PCR results showed that the mRNA levels of METTL3 and ALKBH5 in RA-FLSs were markedly higher than those in OA-FLSs,and the mRNA expression levels of the other two enzymes（METTL14,FTO）were not statistically different between them.2.The m6A modification of Circ 0088194 exists in RA-FLSs,but the relative contents of m6A-modified Circ₀088194 did not differ between RA-FLSs and OA-FLSs.Conclusion:The m6A modification of Circ₀088194 exists in RA-FLSs,but it did not affect the Circ0088194 stability.