| PurposeRheumatoid arthritis(RA)is a chronic systemic autoimmune disease.Disorders of the immune system can cause chronic synovitis and vasospasm,which can cause damage to the synovial membrane,cartilage and bone of the joint,and eventually can cause joint deformity and loss of function.Immune disorders can also stimulate immune cells to produce cytokines and promote synovial inflammation.Some cytokines can increase the expression levels of MMPs by regulating different signaling pathways,directly acting on joint bones and cartilage,causing irreversible damage.Although many drugs have been found to treat this heterogeneous disease,30%of patients still have poor control of the disease.MicroRNAs(miRNAs)are a class of single-stranded small-molecule RNAs that are present in cells.There have been many reports that miRNAs abnormally expressed in RA patients can play an important role in the development of RA by targeting the regulation of signaling pathways and expression of immune molecules in the immune system.Therefore,in this subject,we have studied miR-145-5p as a research object,aiming to study the regulation mechanism of miR-145 on IL-17 in RA.We hope to further understand the pathogenesis of RA,which can provide important clues and new therapeutic targets for the diagnosis,personalized treatment and prognosis of RA.MethodThis experiment was conducted in two cell lines,RA-CD4＋T cells and RA-FLS.The expression level of miR-145-5p in RA-CD4+T cells and other subgroups of T cells was detected;overexpression of miR-145-5p in RA-CD4+T cells,detection of transfection results by Q-PCR;detection of differentiation of Th-17 cells after overexpression of miR-145-5p using flow cytometry;the expression level of IL-17 after overexpression of miR-145-5p was detected using Q-PCR.In RA-FLS,NF-κB,P53/MDM2,JNK and MAPK pathways were added to the corresponding pathway inhibitors BAY11-7082,Nutlin-3,SP600125 and SB203580 in the presence of overexpression of miR-145-5p;select pathways that can significantly inhibit the expression of matrix metalloproteinases for further investigation;Western blot were used to assess changes in the expression levels of key regulators in the signaling pathway;immunofluorescence analysis was used to detect the nuclear localization and activation of key regulatory factors.The expression of matrix metalloproteinase was detected by adding IL-17 stimulation in RA-FLS medium by enzyme-linked immunosorbent assay(ELISA).Result1.The expression level of miR-145-5p in RA-CD4+T cells was higher than that in other subgroups of T cells.2.After overexpression of miR-145-5p in RA-CD4+T cells,the flow assay showed an increase in the proportion of Th-17 cells and an increase in IL-17 expression.3.In the inhibitory experiments of the four pathways,only the chemical inhibitor BAY11-7082 of the nuclear factor-kappaB(NF-κB)signaling pathway significantly attenuated the expression of MMP-9,and no inhibitors of other pathways were observed for MMPs significantly.4.The level of p-P65 was significantly increased after overexpression of miR-145-5p,while the level of IikB-α was significantly decreased.Conversely,a decrease in miR-145-5p resulted in an opposite result.5.Overexpression of miR-145-5p promoted P65 into the nucleus from cytoplasmic entry,which is a marker of activation of the NF-κB pathway.6.IL-17 was added to the RA-FLS medium to stimulate the cells,and the secretion of MMP-9 was increased.ConclusionOverexpression of miR-145-5p significantly promoted the differentiation of Th-17 cells in RA-CD4+T cells and increased the expression level of IL-17.At the same time,miR-145-5p,which is highly expressed in RA-FLS,can promote the expression of MMP-9 by activating NF-κB pathway.IL-17 can also act on RA-FLS cells in a paracrine manner to further promote the expression of MMP-9.Thereby increasing the destruction of the joint bone and cartilage,accelerating the process of bone erosion and increasing the severity of the disease. |
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