| Purpose Rheumatoid arthritis(RA)is a kind of chronic autoimmune disease,which accounts for more than 1% of the world's population.The main features of RA are chronic inflammatory responses and the destruction of articular cartilage and bone,disabling or even inducing other complications.When RA lesions occur,immune disorder caused by abnormal synovial hyperplasia,then generate pannus invasion of cartilage,causing the damage of bone and cartilage,disease heavier joint disease will make the deformity and loss of motor function in patients with.Many cytokines produced by immune disorders play an indispensable role in the complex pathogenesis of RA.Some of these cytokines induce the abnormal proliferation of synovial cells,and then the blood vessels are formed to invade cartilage.Some will increase the production of Matrix metalloproteinase(MMPs)by regulating different signaling pathways,and directly aggravate the damage of articular bone and cartilage.Many studies have shown that miRNAs play a regulatory role in diseases,especially differences.Methods In this experiment,Quantitative PCR(qPCR)was used to measure the expression of mir-145-5p in PBMC of 30 RA patients and 23 normal donors,and the differences of mir-145-5p expression were compared.Meanwhile,the expression differences in synovial tissues of patients with RA(12 cases)and Osteoarthritis(OA)(10 cases)were also measured.The RA patients with RA-Fibroblast-like synovial cells(RA-FLS)were transfected with the miR-145 mimic and control.Q-PCR,Western Blot detection mRNA and protein levels of intracellular Interleukin(IL)-1 beta,IL-6,IL-17 and MMPs(MMP-3 ? MMP-9 and MMP-13)in RA-FLS,again by Enzyme-linked immunosorbent assay(ELISA)experiment analyzed quantity of expression of inflammatory factor,the MMPs in RA-FLS culture supernatant.Then using bioinformatics software and query data analysis to predict potential target genes of miR-145-5p and using qPCR experiment measure the expression of mRNA level,and then using Western blot measure the protein expression,finally using dual luciferase report system to verify the targeted relationship of miR-145-5p and therelated target gene.Results1.The expression of miR-145-5p in PBMC of RA patients was significantly higher than that in PBMC in normal control,and the expression of miR-145-5p in synovial tissue of RA patients and FLS was higher than that in OA patients.2.After overexpression of miR-145-5p,the expression of IL-1?,IL-6 and IL-17 in RA-FLS was detected.The expression of IL-17 was significantly increased,but the expression of IL-1? and IL-6 have no difference.3.After overexpression of miR-145-5p,the expression of MMPs in RA-FLS was determined.It was found that the expression of MMP-3,MMP-9 and MMP-13 increased,and the expression of MMP-1 has no difference.The correlation between IL-17 and MMPs was demonstrated in many RA studies.4.Bioinformatics analysis combined with query data suggested that the untested target genes of mir-145-5p were Ets-1,qPCR and Western blot,which were detected after the mir-145-5p in the RA-FLS,and then decreased after miR-145-5p.5.The dual-luciferase report system verified the targeted relationship between mir-145-5p and Ets-1,and found that mir-145-5p was indeed binding to the 3'UTR region of Ets-1,binding site to: 2710 sites for the 3'UTR of Ets-1.Conclusion The expression of mir-145-5p in PBMC in RA patients was significantly higher than that in normal PBMC.The level of mir-145-5p in the synovial tissues and FLS of RA patients was higher than that in patients with OA.High levels of miR-145-5p of RA may targete the Ets-1 and downregulating its expression,and Ets-1 may increase expression quantity of IL-17,also the increased IL-17 may by adjusting p38 lightning MAP and ERK1/2 channel cause the high level of MMP-3,MMP-9,MMP-13.Therefore,the regulation of cytokines in RA can affect the pathological process of RA. |
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