Mechanisms Of Microrna Regulators FOXP3 And CXCL13 Which Are Related Etiologic Factors Of Myasthenia Gravis

PART1 The preliminary study of Jurkat cells culture and transfection, total RNA extraction and determinationObjective:Jurkat cells were cultured in vitro,miR-125a-5p(miR-548k) mimetics or inhibitors were transfected into jurkat cells.Total RNA was extracted and fully prepared for the later experiment.Methods:Cultured jurkat cells(106/ml) with RPMI 1640 medium were transfected in 6-well plates transfected with miR-125a-5p(or miR-548k) mimic, mimic negative control, miR-125a-5p (or miR-548k) inhibitor or inhibitor negative control and further incubated for 48 h. Total RNA was prepared from jurkat cells using Trizol.Results:Jurkat cells suspended in medium and hold together.They grow fast and subculture after 2 to 3 days. At the 4 to 5 generations later, cells can be used for subsequent experiments.Cells grow in good condition after transfection.Totol RNA was extracted in good concentration and quality.Conclusion:1. Jurkat cells were suitable for further research in this experiment.2.Total RNA was extracted appropriate to subsequent experiments in this study.PART 2 Mechanisms of miR-125a-5p downregulates the expression of Foxp3 in jurkat cellsObjective:The aim of this study is to explore the miR-125a-5p regulation of CXCL13 after transfected with mimic and inhibitor of miR-125a-5p by using the QRT-PCR and Western Blotting.Methods:Detection of the relative expression of miR-125a-5p and Foxp3 mRNA by quantitative real-time PCR(QRT-PCR). Relative expression level of the protein Foxp3 measured by Western Blotting.Results:When Jurkat cells were transfected with miR-125a-5p mimics,it was observed that the expression of miR-125a-5p increased obviously, however the expression of Foxp3 mRNA and the protein Foxp3 were decreased. In contrast, transfection with miR-125a-5p inhibitor rduced the expression of miR-125a-5p and increased the expression of Foxp3 mRNA and the protein Foxp3.Conclusion:1.In Jurkat cells miR-125a-5p modulated Foxp3 expression after transcription.2.MiR-125a-5p inhibits Foxp3 expression by post-transcriptional gene silencing.PART 3 Mechanisms of MicroRNA Regulators Foxp3 and CXCL13 which are Related Etiologic Factors of Myasthenia GravisObjective:The aim of this study is to explore the miR-548k regulation of CXCL13 after transfected with mimic and inhibitor of miR-548k by using the QRT-PCR and ELISA.Methods:Detection of the relative expression of miR-548k and CXCL13 mRNA by quantitative real-time PCR(QRT-PCR). Relative expression level of the protein CXCL13 measured by ELISA.Results:.When Jurkat cells were transfected with miR-548k mimics, the expression of miR-548k increased obviously,however the expression of CXCL13 mRNA and the protein CXCL13 were decreased. In contrast, transfection with miR-548k inhibitor rduced the expression of miR-548k and increased the expression of CXCL13 mRNA and the protein CXCL13.Conclusion:1.In Jurkat cells miR-548k regulated CXCL13 expression after transcription.2.MiR-548k inhibits CXCL13 expression by post-transcriptional gene silencing.