| Research Background:Rheumatoid arthritis(RA)is a chronic systemic autoimmune disease characterized by chronic synovitis.The FLS plays a key role in the pathogenesis of RA.They are "persistently activated cells" with characteristics similar to those of tumor cells,which may exhibit excessive proliferation,suppressed apoptosis,and a significant increase in migration and invasion,and are key factors in joint destruction and inflammation,although the mechanisms underlying their altered biological behavior are unclear.Recent studies have shown that high expression of TGFβ1 cytokines in the joints of RA patients can promote epithelial-mesenchymal transition(EMT)in RA-FLS to promote synovial inflammation.Long noncoding RNA(lncRNA)is a class of RNA molecules that are more than 200 nt in length and do not have protein-coding ability,which are abundant and widely distributed in the body of organisms.Recent studies have shown that lncRNAs are associated with the proliferation and apoptosis of RAFLS cells and other pathological processes.In this study,we intend to investigate the expression and function of LncRNA in TGFβ1-induced EMT transformation of RA-FLS cells,and to explore its regulatory mechanism in the inflammatory process of RA synovium.Part I.Screening of TGF-β1 pathway-related LncRNA in knee synovial-like fibroblasts Objective1.Detection of LncRNA expression profiles in RA-FLS and control group after TGF-β1 stimulation to screen for differentially expressed lncRNAs2.Observation of expression and function of ITGB8-AS1 in RA-FLS cells cultured in vitroMethodsSynovial tissues of knee joints from RA patients were obtained from knee replacement surgery and knee arthroscopic examination,and FLS cells were cultured in vitro using the tissue block apposition method.Total cellular RNA was extracted from three RA-FLS and three TGF-β1-stimulated RA-FLS cases,and IncRNA expression profiles were detected by LncRNA Array microarray to obtain differentially expressed IncRNAs in RA-FLS and TGF-β1-stimulated RA-FLS.The differentially expressed lncRNAs screened by the microarray were detected and validated using RT-qPCR,and the IncRNAs that appeared to be stably differentially expressed were selected as the study target IncRNAs.IncRNA ITGB8-AS1 was tested for its function in RA-FLS cultured in vitro by EdU,CCK8,in vitro invasion and migration assays.ResultsVimentin and Cadherin-11 protein expression was detected in primary cells isolated and cultured in vitro and identified as RA-FLS cells.IncRNA microarray results yielded 84 LncRNAs differentially expressed in RA-FLS and RA-FLS after TGF-β1 stimulation treatment(Fold change>2-fold),of which 84 LncRNAs were differentially expressed in RA-FLS after TGF-β1 stimulation-treated RA-FLS had 47 LncRNAs with up-regulated expression and 37 with down-regulated expression.LncRNA ITGB8-AS1,which was significantly down-regulated in RA-FLS,was selected for the study.Compared with RA-FLS after TGF-β1 stimulation treatment,LncRNA ITGB8-AS1 showed stable expression down-regulation after TGF-β1 stimulation treatment in the other five RA-FLS cultured in vitro.By overexpressing LncRNA ITGB8-AS1 in RA-FLS using adenovirus and after interfering with the expression of LncRNA ITGB8-AS1 using siRNA,it was found that LncRNA ITGB8-AS1 could inhibit its proliferation,invasion and migration ability in RA-FLS,but had no effect on its apoptosis and cell cycle.ConclusionCompared with RA-FLS,LncRNA expression profile appeared significantly altered in RA-FLS after TGF-β1 stimulation treatment.ITGB8-AS1 expression was down-regulated in RA-FLS cultured in vitro from RA patients,and it could inhibit the proliferation,invasion and migration ability of RA-FLS cultured in vitro,but had no effect on its apoptosis and cell cycle.Part Ⅱ Long-stranded non-coding RNA ITGB8-AS1 regulates inflammation in CIA mice knee joint synovial tissueObjective1.To study the effect of ITGB8-AS 1 on knee joint synovial inflammation in CIA mice2.To explore the mechanism of ITGB8-AS1 regulation of knee joint synovial inflammation MethodsThe group demonstrated that LncRNA ITGB8-AS1 can bind to protein FLNA and used RIP to verify the binding in the synovial tissue of CIA mice.The collagen-induced arthritis(CIA)mouse model was constructed,and the mice were injected with type 5 adeno-associated virus overexpressing LncRNA into the knee joint cavity to specifically express ITGB8-AS1 in the synovial tissue of the knee joint.The development of inflammatory response in mice was monitored.After decalcification,paraffin-embedded mice knee joint tissues were observed for synovial inflammation.Assess the role of LncRNA in mice in vivo.To detect protein expression levels by immunoblot(Western blot)assay and immunohistochemistry,and to detect RAC 1 protein activity in mouse knee synovial tissue proteins using G-LISA method.ResultsIn the CIA mouse model,overexpression of ITGB8-AS1 reduced the inflammatory response in the synovial tissue of the knee joint,and the proliferation of synovial tissue was inhibited in paraffin sections compared with the control group.lncRNA ITGB8-AS1 could bind to protein FLNA and inhibit the binding and activation of FLNA to small GTPase RAC1.lncRNA ITGB8-AS1 overexpression could bind to protein FLNA and inhibit the binding of FLNA to small GTPase RAC1.LncRNA ITGB8-AS1 overexpression inhibited RAC1 activity via FLNA and downregulated N-Cadherin vimentin and a-SMA protein expression levels.It inhibited the epithelial mesenchymal transition of FLS cells and attenuated the inflammatory response in the synovial tissue of knee joints of CIA mice.ConclusionThe upregulation of LncRNA ITGB8-AS1 expression in the knee joint of CIA mice resulted in reduced synovial tissue proliferation,reduced angiogenesis,and reduced lymphocyte infiltration,as well as reduced cartilage destruction.LncRNA ITGB8-AS1 binds to FLNA protein to inhibit the activation of RAC 1 by FLNA,which in turn regulates the epithelial-mesenchymal transition of synovial-like fibroblasts The LncRNA ITGB8-AS1 was shown to exert an inhibitory effect on the synovial inflammatory response. |
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