The Effect And Mechanism Of EphB2 In B Cell Activation

Part 1 A preliminary study about the expression and effect of EphB2 on B cellsBackground:EphB2 is a member of Eph receptor tyrosine kinase family, primarily expresses on tumor cells and endothelial cells, can induce proliferation and migration of tumor cells and promote endothelial cell cluster. Recent studies have found that EphB2 also expresses on T cells and monocytes, moreover, ligand-induced forward EphB2 signaling can promote T cell migration and monocyte activation. However, the expression and effect of EphB2 in B cell activation is still unclear.Methods:Venous blood of healthy volunteers were extracted and sorted B lymphocytes. Naive B cells were activated by stimulating with LPS, anti-IgM and IL-4 respectively, then the expression of EphB2 and its ligand ephrinB1/B2 proteins were detected by western blot. EphB2 siRNA was transfected into B cells to inhibit EphB2 expression, at the same time, negative control siRNA (NC) transfected group and non-transfected group were set up, and B cell proliferation, secretion of cytokines TNF-α and IgG/IgM antibody levels were detected.Results:EphB2 and its specific ligand ephrinB1/B2 expressed on human peripheral blood naive B cells, after stimulating B cell activation in vitro, the expression of EphB2 significantly increased (P<0.05), while the expression of ephrinBl and ephrinB2 did not change significantly. At the same time, we transfected EphB2 siRNA to inhibit EphB2 expression on B cells and detected the markers of B cell activation, the results showed that B cell proliferation (P<0.05), TNF-α secretion (P<0.01) and IgG production (P<0.05) were decreased significantly in EphB2 siRNA group compared with siRNA NC and non-transfected control groups. While, the levels of IgM in all groups had no difference.Conclusions:EphB2 and its ligand ephrinB1/B2 express on human B cells, and EphB2 plays an important role in promoting B cell activation.Part 2 Screening and identification of microRNAs that can regulate EphB2 expression on B cellsBackground:microRNAs are a class of endogenous,20-to 24-nucleotides small RNA, can specifically bind to and degrade mRNA or inhibit it to translate into protein, and have become the hotspot of disease’s molecular targeted therapy for their stability, conservative feature and targeting binding specificity. Studies have shown that the expression of EphB2 can be regulated by a variety of microRNAs in glioma cells and vascular endothelial cells. Therefore, screening for a microRNA that can specifically regulate EphB2 expression on B cells has an important value for the further studies about the mechanisms of EphB2 effects on B cells.Methods:Using miRWalk database and comprehensive referencing five bioinformatics analytical methods, microRNAs that can regulate the expression of human EphB2 were screened and their expressions in B cell were detected using quantitative real-time PCR. Thereafter, luciferase reporter plasmids that contain binding sites of EphB2 3’UTR were constructed, and were co-transfected with miR-185 mimics or mimics NC into the 293 T cells, then the fluorescence of cells in each group was measured in order to verify whether miR-185 can specifically bind to EphB2 mRNA and affect EphB2 expression. In addition, after transfecting miR-185 mimics and inhibitors into the B cells, the expression of EphB2 protein was detected by western blot, the percentage and the mean fluorescence intensity of EphB2 (+) B cells were measured by flow cytometry in order to further verify whether miR-185 can regulate the expression of EphB2 on B cells; at the same time, B cell proliferation, secretion of TNF-α and IgG/IgM antibody were detected to explore the influence of miR-185 on B cells activation.Results:miRWalk database screened miRNA-185, miRNA-661, miRNA-593, miRNA-211, miRNA-515 and miRNA-204 can regulate the expression of human EphB2. These six microRNAs all expressed in human peripheral blood naive B cells, but only the levels of miRNA-185, miRNA-515 and miRNA-204 down-regulated (P <0.05) in activated B cells, and compared with miRNA-515 and miRNA-204 expression, miRNA-185 expression decreased more obviously (P<0.05). Luciferase assay showed that the value of fluorescence activity in the wild type binding sites of EphB2 3’UTR and miRNA-185 mimics co-transfected group decreased by 41%(P <0.01). Further, after we respectively transfected miR-185 mimics and inhibitors into B cells, and compared with the NC group and the non-transfected control group, the expression of EphB2 on B cells, the percentage and the mean fluorescence intensity of EphB2 (+) B cells were significantly reduced (P<0.05) in miR-185 mimics transfected group, while, the expression of EphB2 on B cells, the percentage and the mean fluorescence intensity of EphB2 (+) B cells were significantly upregulated (P<0.05) in miR-185 inhibitor group. At the same time, we measured the markers of B cell activation, the results showed that B cell proliferation (P<0.05), the levels of TNF-α (P<0.01) and IgG (P<0.05) in B cell culture supernatant were significantly decreased in miR-185 mimics transfected group, and B cell proliferation (P<0.05), the levels of TNF-α (P<0.01) and IgG (P<0.05) in B cell culture supernatant were significantly increased in miR-185 inhibitor transfected group.Conclusions:miR-185 can depress B cell activation through specifically inhibiting the expression of EphB2.Part 3 The molecular mechanisms of EphB2 regulating B cell activationBackground:NF-κB is the core transcription factor in cell activation, and p65, a subunit of NF-κB, is an important signaling molecules in B cell activation. In nerve cells, Src is an important signaling molecules at the downstream of EphB2, can promote the formation and remodeling of synapse. Recent studies indicate that the activation of Src can activate NF-κB (p65) molecules. We speculate that EphB2 may regulate B cell activation through Src-p65 signaling pathway. In addition, Notch1, a recently discovered signaling molecules, is involved in T cell activation, but its role in B cell activation is not clear. In this part we will explore the role of these signaling molecules in the process of EphB2 regulating B cell activation.Methods:EphB2 siRNA or miR-185 mimics were transfected into B cells to inhibit EphB2 expression, and western blot was performed to detect phospho-Src/total Src, phospho-p65/total p65 and Notch1 protein levels in B cell, as to explore the signaling pathways in EphB2 regulation B cell activation.Results:Compared with the control groups, the level of phosphor-Src was significantly reduced (P<0.05) while the level of total Src showed no significant difference in the EphB2 siRNA and miR-185 mimics transfected groups; and the level of phosphor-p65 was obviousely decreased (P<0.05) while the level of total p65 showed no significant difference in the EphB2 siRNA and miR-185 mimics transfected groups. Meanwhile, the expression of Notch1 was also significantly inhibited in the EphB2 siRNA and miR-185 mimics transfected groups (P<0.05).Conclusions:EphB2 may regulate B cell activation through Src-p65 signaling pathway and Notch1 is also play an important role in it.