Effects Of MicroRNA-146a On Myasthenia Gravis

Objective:The aim of our study was to identify the expression of miRNA-146a in peripheral blood mononuclear cells (PBMCs) obtained from patients with myasthenia gravis (MG) and correlate these changes with healthy controls; to investigate the effect of inhibition of miR-146a on the activation of acetylcholine receptors-specific B cells obtained from mice; to explore the role of miR-146a in the pathogenesis of MG and look forward to the new therapy methods in MG.Methods:The expression of miRNA-146a in PBMCs obtained from patients with MG and healthy controls were determined by miRNA qRT-PCR. MiR-146a’s complementary fragment named antagomiR-146a was synthesised as inhibitor, and the nonfunction fragment, which has similar construction to antagomiR-146a was synthesised as control. Magnetic active cell sorting (MACS) was used to isolate B cells and T cells in the spleen of mice before identified these cell’s purity and cytoactive; Transfect the HiPerFect contain FITC-antagomiR-146a into B cells and detect the uptake rate of antagomiR-146a by flow cytometry. The isolated T cells and B cells were co-cultured in the ratio1:3, and these cells were randomly divided into four groups:blank group, Tα group, Tα+inhibitor group and Tα+control group.2days after co-cultured, Tα were added into all groups except the blank group, meanwhile, antagomiR-146a and control fragment were given to Tα+inhibitor group and Tα+control group as intervenor for3days. At the end of the intervention, B cells were sort by MACS. MiRNA qRT-PCR was used to detect the expression of miR-146a in B cells; flow cytometry was used to analyze the expression of CD40, CD80and CD86on the surface of B cells; Western blot was used to detect the expression of TLR4, NF-κB and Bcl-2.Results:1.Our findings demonstrated that the expression of miRNA-146a in PBMCs obtained from patients with MG was significantly upregulated compared to healthy controls (p=0.004). 2. The purity of isolated B cells and T cells were97.1%and96.7%; the cytoactive of B cells verified by3H-TdR incorporation certified was within the normal range; the uptake rate of antagomiR-146a was65.8%; these elements are good enough to meet the experimental requirements.3. The expression of miR-146a in B cells from Ta group was significantly upregulated compared with blank group (p<0.001), and it from Ta+inhibitor group was significantly downregulated compared with Ta group (p<0.001). But there was no significant difference between Ta group and Ta+control group (p=0.948).4. In Ta group, The expression of CD40, CD80and CD86on the surface of B cells was significantly upregulated compared with blank group, Whereas in Ta+inhibitor group the expression of CD40and CD80were significantly downregulated compared with Ta group. There were no significant differences between the expression of CD86in Ta+inhibitor group and CD40, CD80, CD86in Ta+control to Ta group.5. The expression of TLR4and NF-κB in B cells from Ta group was significantly upregulated compared with blank group (p＜0.001), and these from Ta+inhibitor group were significantly downregulated compared with Ta group (p<0.001). But there were no significant difference between Ta group and Ta+control group. There was no significant difference in expression of Bcl-2in all the goups (p>0.05).Conclusion:1. MiR-146a was significantly upregulated in the PBMCs of MG patients compared to healthy controls.2. MiR-146a may play an important role in the regulation of the activation of B cells and can be involved in the pathogenesis of MG.