The Effect Of MicroRNA-20b On T Cells Of Thymoma-associated Myasthenia Gravis Via NFAT Signaling Pathway

Objective:In this study,we aimed to evaluate the effects and mechanism of miR-20 b on T cells in thymoma-associated myasthenia gravis.Methods:1.The relative miR-20 b expression levels in thymoma and normal thymus were studied by RT-PCR.2.We raised the expression of miR-20 b by transfecting the cells with miR-20 b mimics in Jurkat cells.Then we analyzed the effects of microRNA-20 b on T cell proliferation and activity by MTT method.3.We used Target Scan and miRanda to predict potential effectors of mi R-20 b.Then confirm our forecast by RT-PCR and Western Blot.4.We analyzed the effects of target genes on T cell proliferation and activity by MTT method.5.RT-PCR and Western Blot were used to determine the relation of miR-20 b and target genes in thymoma.6.The expression levels of NFAT5 and CAMTA1 were analyzed respectively in Jurkat cells co-transfecting with miR-20 b mimics or anti-mi R-20 b and si-NFAT5 or si-CAMTA1.Finally,mechanism studies were conducted by knocking out the target gene.Results:1.We examined the levels of miR-20 b in serum from myasthenia gravis patients with or without concurrent thymoma,the results showed that levels of miR-20 b were lower in myasthenia gravis patients compared to healthy participants.Interestingly,miR-20 b levels were even lower in gravis patients with thymoma than those without thymoma.2.Expression levels of miR-20 b were significantly downregulated in activated T cells,including activated Jurkat cell and CD4+T cell.Increased miR-20 b expression induced G0/G1 arrest and decreased miR-20 b expression relieved G0/G1 arrest and promoted cell cycle progression.3.The expression of target genes showed down-regulation with transfecting miR-20 b mimics,and up-regulation with transfecting anti-miR-20 b.4.The expression of IL2?IL10?CD25 and CD69 decreasedwith transfecting miR-20 b mimics,which means that the activity of cell proliferation was inhibited,correspondingly the results increased with transfecting anti-miR-20 b.5.MiR-20 b was negatively correlated with the target genes in thymoma.6.Jurkat cells psuppress T cell proliferation and activation after silencing target genes,the function can be partially offset by decreasing the expression of miR-20 b.These results demonstrated that NFAT5 and CAMTA1 were the target genes of miR-20 b in thymoma with myasthenia gravis.Conclusion:As a negative regulation factor,miR-20 b inhibits T cell proliferation and activity in thymoma-associated myasthenia gravis.Mi R-20 b plays an important immunoregulatory role by the regulation of NFAT5 and CAMTA1 gene in thymoma-associated myasthenia gravis.MiR-20 b can be used as a potential marker in thymoma-associated myasthenia gravis.