Research Of T Helper 17 Cells And Its Regulatory Mechanisms In Myasthenia Gravis Related Thymomas

Part one: Th17 cells and their associated cytokines in thymomas from patients with myasthenia gravis.Objective: Myasthenia gravis (MG) is a CD4+ T cell dependent autoimmune disease mediated by anti-acetylcholine receptor autoantibodies (AchR-Abs) at the neuro-muscular junction. The role of Th17 cells in autoimmune MG development,pathogenesis and prognosis are not well illustrated. The purpose of this study is to explore whether or not Th17 cells and their related cytokines are altered in MG patients with thymomas.Methods: Fifty-seven myasthenia gravis (MG) patients were divided into two groups: MG with thymomas (TM) group (n=35),and MG with thymic hyperplasia (TH)group (n=22). The healthy controls (HC) included 22 volunteers. Th17 cells from peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry. The mRNA expression levels of cytokines in PBMCs were determined with real-time quantitative-polymerase chain reaction (RT-PCR) . Protein levels of Thl7 related cytokines were detected with enzyme-linked immunosorbent assay (ELISA).Results: The quantity of Th17 cells in TM group was significantly increased compared with that in healthy control (P<0.05) while there was no significant differences between TH group and HC group (P>0.05). The mRNA and protein levels of IL-17, IL-1 ?, IL-6 and IL-23 in TM group were up-regulated significantly versus those in HC group(P<0.05).Meanwhile, the expression levels of IL-1? proteins in TM group were up-regulated significantly versus those in HC group (P<0.05) .However,there were no significant differences in IL-17, IL-6 and IL-23 mRNA and proteins between TH group and HC group (P>0.05).Conclusion: The elevated levels of IL-17 and Th17 related cytokines in thymomas may aggravate the autoimmunity disorders.Part two: MicroRNA-19b-mediated epigenetic regulation in the pathogenesis of myasthenia gravis with thymomasObjective Th17 cells are required in the pathogenesis of MG with thymomas.However the underlying mechanism for Th17 cells generation in MG with thymoma is still greatly unknown. The role of microRNA in thymoma development and their pathogenesis are not well illustrated. Thymic stromal lymphopoietin (TSLP) acts directly on Thl7 cells by reducing their capacity to produce interleukin (IL)-17 and fostering the development of Th17 cells. The purpose of this study is to explore that microRNA-19b-5p- related-pathway regulates the decrease expression levels of TSLP in vitro and the regulatory mechanisms of miRNA-19b-5p in T helper cells differention in MG patients with thymomas.Methods In total, 52 MG patients with thymomas (MG-T group), 12 thymomas patients without MG (NMG-T group) and 11 healthy controls (HC group) were enrolled from the Department of neurology of the 309 Hospital of Chinese People's Liberation Army. The mRNA expression levels of TSLP and microRNA-19b in thymomas were detected by RT-PCR. The protein levels of TSLP in thymomas were detected by Western blot. Spearman test was used for correlation analysis between microRNA-19b and TSLP mRNA or protein levels. We engineered luciferase reporters to confirm the possibility that TSLP were directly targeted by miR-19b-5p.Results The expression values of TSLP mRNA in MG-T group and in NMG-T group were significantly lower than those in normal thymus. Additionally, the expression of TSLP mRNA was significant different between thymoma patients with and without MG(P<0.05 ). There were significant differences between thymoma of type B1 (0.58±0.08) and thymoma of type B2 or thymoma of type B3. Meanwhile, the expression levels of mature miR-19b-5p in thymomas were more remarkably increased than those in healthy subjects. Using Pearson's correlation coefficient analysis, we found that there was a significant negative correlation between the expression level of TSLP mRNA and the expression level of mature miR-19b-5p in MG-T patients. By luciferase reporter assay, we proved that microRNA miR-19b-5p directly targets TSLP mRNA 3'UTR and regulates TSLP expression.Conclusions These results indicated that the elevation of microRNA-19b in thymomas suppressed TSLP expression and then influenced Th17 cells development in thymomatous MG. Our findings suggest that microRNA-19b-5p-related-pathway might be involved in the pathogenesis of MG.