Study On The Role And Mechanism Of WTAP-Mediated LncRNA CRNDE M~6A Modification In Rheumatoid Arthritis

BackgroundRheumatoid arthritis(RA)is a common autoimmune disease,which is mainly characterized by persistent synovial inflammation of joints.In the later stage of the disease,it will lead to the destruction of articular cartilage and bone,even the dysfunction of joint function,which will lead to disability,seriously affecting patients’the quality of life.During the inflammatory process of RA,the proliferation and migration and invasion ability of fibroblastoid synovial cells(FLS)are the king factors to relieving inflammation,making an ideal target of anti-RA.Studies showed that LncRNA expression changes in FLS and plays an important role in the progression of RA inflammation.LncRNA CRNDE has been reported to have proinflammatory effects in a variety of inflammation-related diseases,but its role and regulatory mechanism in RA remain to be explored.At the same time,FLS can undergo phenotypic changes through epigenetics in the inflammatory process of RA,and m~6A,as a common post-transcriptional modification,plays an important role in RA.Therefore,exploring the molecular mechanism of m~6A methylation regulation of LncRNA CRNDE expression may provide more ideas for the treatment of RA.In colorectal cancer,LncRNA CRNDE can inhibit DUSP5 expression by binding to EZH2 and play a role in promoting cancer.DUSP5 has been reported to have important anti-inflammaion effects in RA.DUSP5 can alleviate RA inflammation by inhibiting NF-κB signaling pathway,and reduce the expression of pro-inflammatory factors and clinical severity of arthritis in RA mouse models.Therefore,LncRNA CRNDE may regulate the signaling pathway and promote the progression of inflammation by inhibiting DUSP5expression in RA FLS.SO,the study on the role and mechanism of WTAP-mediated methylation modification of LncRNA CRNDE m~6A in rheumatoid arthritis was carried out.ObjectiveThis study aims to explore the upstream and downstream the molecular mechanisms of CRNDE’s effects on RA FLS,The Exploration the role of LncRNA CRNDE in CIA,in order to provide a new target for RA treatment.Methods1.To observe the role of LncRNA CRNDE in RA,the CIA mouse inflammation model was constructed by subcutaneous injection of Chicken typeⅡcollagen into the tail root of DBA/1J male mice.The CIA mouse inflammatory model was treated by local injection of PBS,LV-sh-NC and LV-sh-CRNDE.Physiological data such as the degree of swelling,thickness and weight change of the paw were measured by vernier calipers and weight scales to monitor the inflammatory process of CIA mice.H&E staining and saffron solid green staining were used to observe the pathological characteristics of the plantar joint of mice after treatment.2.In order to observe cell proliferation,migration and invasion,CRNDE,WTAP and DUSP5 were overexpressed and silenced by Lipofectamine 3000 kit.CCK-8 reagent and Transwell assay were used to detect cell proliferation,migration and invasion.Migratory and invasive samples were photographed using an inverted fluorescence microscope at 20 x objective magnification.3.MH7A cells were treated with different concentrations of TNF-α,and RNA was extracted 24 hours later to observe the expression of CRNDE,WTAP and DUSP5 by RT-qPCR.48 hours later,cell proteins were extracted and the expressions of WTAP,DUSP5,ERK1/2,p-ERK1/2 and other proteins were detected by WB assay.4.The expression position of CRNDE was observed by fluorescence probe localization.5.After WTAP was overexpressed and silenced by Lip3000 for 24 h,cells were treated with Actinicin-D,and cell RNA was extracted at 0 h,2 h,4 h,6 h,and 8 h.CRNDE expression was detected by RT-qPCR,and then the half-life of CRNDE was calculated.6.To detect the m~6A methylation level of RNA,The m~6A methylation test kit was used to detect the m~6A methylation modification level,and the m~6A spot hybridization test was used to detect the total RNA The level of m~6A methylation modification,the prediction of m~6A methylation of CRNDE using the SRAMP database website,the detection of m~6A modification of CRNDE after inflammatory stimulation by me RIP-qPCR assay and the binding ability of methyltransferase WTAP to CRNDE by RIP assay.7.From the PAWS of mice treated with normal NC group and CIA model group,the expression of CRNDE was observed by RT-qPCR method.After histopin was extracted,the expression of WTAP,DUSP5,ERK1/2,p-ERK1/2 and other proteins was detected by WB.The expression of DUSP5 was detected by immunohistochemistry.Results1.In RA animal model CIA mice,CRNDE expression was significantly increased,and lentivirus silencing of CRNDE could significantly relieve the inflammatory process and reduce synovial inflammation and cartilage erosion.The expression of CRNDE was up-regulated in RA FLS cells,and its expression level was positively correlated with the proliferation,migration and invasion of FLS cells.2.In MH7A cells,m~6A methylation modification was significantly enhanced after TNF-αstimulation,and the expression of methyltransferase WTAP was increased.WTAP was positively correlated with m~6A methylation modification,cell proliferation,migration,and invasion of MH7A cells as a whole.3.WTAP can promote CRNDE expression through m~6A methylation modification,and promoting the proliferation,migration and invasion of MH7A cells.4.The expression of DUSP5 in CIA is significantly decreased,and it promotes the proliferation,migration and invasion of MH7A cells by up-regulating the phosphorylation of ERK1/2,that is,there is DUSP5-ERK1/2 pathway in MH7A cells.5.In CIA,CRNDE regulates ERK1/2 pathway by inhibiting DUSP5,and WTAP affect DUSP5-ERK1/2 pathway by stabilizing CRNDE to regulate the biological function of FLS.ConclusionIn this study,by exploring the role of CRNDE in CIA mouse inflammatory model and MH7A cells and its molecular regulatory mechanism,it was revealed that WTAP promoted the expression of CRNDE and inhibited the expression of DUSP5 in RA FLS through m~6A modification.Furthermore,it promotes the proliferation,migration and invasion of RA FLS cells mediated by ERK1/2 pathway activation to regulate the inflammatory process.The results suggest that"CRNDE"can provide a new scientific basis for anti-RA.