Hsa＿circ＿0088036/miR-140-3p/SIRT1 Axis Promotes Proliferation And Migration Of Rheumatoid Fibroblast-like Synoviocytes

Background:Rheumatoid arthritis(RA)is an autoimmune disease mainly manifested by symmetric inflammatory arthritis.The pathogenesis of RA has not been fully clarified yet.It is clear that during the development of RA,abnormal hyperplasia,migration and invasion of synoviocyte are important pathological changes in the pathogenesis of RA.Circular RNAs(circRNAs)are a new type of non-coding RNAs with stable,high expression and tissue specificity.In recent years,the biological function of circRNAs has become a research hot spot.The biological function of circRNAs has been reported mainly in tumor diseases,but few in autoimmune diseases.Our previous study found that the expression of hsa＿circ＿0088036 in peripheral blood mononuclear cells of RA patients was increased.In order to determine the role of hsa＿circ＿0088036 in the pathogenesis of RA,we used bioinformatics to predict that hsa＿circ＿0088036 may promote the proliferation and migration of RA synovial-fibroblasts by regulating miRNA-140-3p/SIRT1.This study was divided into three parts to investigate the mechanism of hsa＿circ＿0088036/miR-140-3P/SIRT1 axis promoting the proliferation and migration of RA synovial-fibroblasts.Part Ⅰ Expression of hsa＿circ 0088036,miR-140-3p and SIRT1 in RA-FLSObjective:To isolate and purify the primary synovial fibroblasts and to detect the expression levels of hsa＿circ＿0088036,miR-140-3p and SIRT1 in synovial fibroblasts.Method:1.Synovial tissues obtained from RA and OA patients in knee replacement surgery were mechanically separated,placed in the cell culture bottle to adherent culture twice,and putified and observed cell morphology by inverted microscope.The cells were identified by immunocytochemistry.2.The expression levels of hsa＿circ＿0088036,miR-140-3p and SIRT1 in RA and OA synovial fibroblasts were detected by RT-PCR.Results:1.Under the inverted microscope,the primary synovial cells were spindle shaped,and more than 98%were fibroblasts.The nuclei were oval and located in the center of the cells.A few round synovial macrophages are occasionally seen.Immunofluorescence staining showed that more than 98%of synovial cells were positive for vimentin.2.Compared with OA,the expression levels of hsa＿circ＿0088036 and SIRT1 in RA synovial fibroblasts were increased,while the expression of miR-140-3p was decreased.Conclusion:1.Human synovial fibroblasts were successfully isolated and purified to obtain high purity,which laid a foundation for subsequent studies on synovial mechanism of rheumatoid arthritis.2.Compared with OA,the expression of hsa＿circ＿0088036 and SIRT1 in RA synovial fibroblasts was abnormally increased,while the expression of miR-140-3p was decreased.Part Ⅱ The effects of hsa＿circ＿0088036 and miR-140-3p on the proliferation and migration of RA-FLSObjective:To determine the effects of hsa＿circ＿0088036 and miR-140-3p on the proliferation and migration of RA synovial fibroblasts by in vitro experiments.Method:1.According to the sequence information of hsa＿circ＿0088036,plasmid construction and adenovirus packaging were performed.RA synovial fibroblasts were transfected with adenovirus containing the hsa＿circ 0088036 plasmid,and the overexpression efficiency was detected by RT-PCR.2.According to the sequence information of hsa＿circ＿0088036,three siRNA targets were designed and transfected into RA synovial fibroblasts.The interference efficiency was detected by RT-PCR,and the siRNA with the highest interference efficiency was selected.3.Mimic and inhibitor of miR-140-3p were constructed and transfected into RA synovial fibroblasts,respectively.Overexpression and interference efficiency were detected by RT-PCR.4.Functional experiment:Overexpression/interference of hsa＿circ＿0088036/miR-140-3p was performed.The proliferation of synovial fibroblasts was detected using EdU method,and the migration by Transwell method.Results:1.The adenovirus transfection method can effectively overexpress hsa＿circ＿0088036,and siRNA can effectively interfere the expression of hsa＿circ＿0088036.2.In RA synovial fibroblasts,the overexpression of hsa＿circ＿0088036 can promote cell proliferation and migration,and the interference of hsa＿circ＿0088036 can inhibit cell proliferation and migration.3.Mimic transfection method can effectively overexpress miR-140-3p,while inhibitor transfection can effectively interfere the expression of miR-140-3p.4.In RA synovial fibroblasts,overexpression of miRNA-140-3p can inhibit cell proliferation and migration,and interference with miR-140-3p can promote cell proliferation and migration.Conclusion:Hsa＿circ＿0088036 can promote the proliferation and migration of RA synovial fibroblasts,while miR-140-3p inhibited the proliferation and migration of RA synovial fibroblasts.Part Ⅲ hsa＿circ＿0088036 regulates the proliferation and migration of RA-FLS by sponging miR-140-3p and affecting the expression of SIRT1Objective:To explore the interaction mechanism of hsa＿circ＿0088036,miR-140-3p and SIRT1.Method:1.Bioinformatics analysis predicted the miRNAs most likely to have binding sites with hsa＿circ＿0088036.Overexpressed hsa＿circ＿0088036 in RA synovial fibroblasts,and screened out the miRNA with the most obvious down-regulated expression,and predicted the downstream target genes of the miRNA through bioinformatics.2.Overexpressed or interfered hsa＿circ＿0088036 in RA synovial fibroblasts,and the expression of miR-140-3p was detected by RT-PCR,the expression of SIRT1 was detected by RT-PCR and Western blot.3.According to the binding site information of hsa＿circ＿0088036 and miR-140-3p,the mutant sequence plasmid was constructed,and the targeted binding relationship between the two was verified by luciferase reporter gene assay.According to the binding site information of miR-140-3p and SIRT1,the mutant sequence plasmid was constructed,and the target binding relationship between the two was verified by luciferase reporter gene assay.4.Rescue experiment:hsa＿circ＿0088036 and miR-140-3p were overexpressed simultaneously in RA synovial fibroblasts to detect the recovery of cell proliferation and migration function.Results:1.Hsa＿circ＿0088036 was overexpressed in synovial fibroblasts of RA,and the expression levels of miR-140-3p,miR-873-5p,miR-611 and miR-612 were all decreased,among which the decrease of miR-140-3p was most obvious.2.Overexpression of hsa＿circ＿0088036 can cause the mRNA level of miR-140-3p to decrease,and the mRNA and protein levels of SIRT1 to increase;Interfering hsa＿circ＿0088036 could increase the mRNA level of miR-140-3p,and decrease the mRNA and protein levels of SIRT1.3.Dual-luciferase reporter gene assay confirmed that hsa＿circ＿0088036 interacted with miR-140-3p,and miR-140-3p interacted with SIRT1.4.The simultaneous overexpression of hsa＿circ＿0088036 and miR-140-3p reversed the regulation of hsa＿circ＿0088036 on the proliferation and migration of synovial fibroblasts in rescue experiments.Conclusion:1.hsa＿circ＿0088036 has binding sites with a variety of miRNAs,in which miR-140-3p is one of its possible target miRNAs.hsa＿circ＿0088036 may exert competitive inhibition to regulate the expression of miR-140-3p through the mechanism of sponge and up-regulate the expression of SIRT1.2.MiR-140-3p has a targeted binding relationship with hsa＿circ＿0088036,and hsa＿circ＿0088036 affects the expression of SIRT1 to regulate the proliferation and migration of RA synovial fibroblasts by sponging miR-140-3p.