The Role And Mechanism Of MiR-130b In B Cell Activation

MicroRNAs(miRNAs)are noncoding RNA,which play important roles in cell proliferation,apoptosis,chronic inflammatory regulation,tumor development and other biological processes.The main function mode of miRNAs is to prevent the translation of mRNA into protein by binding to the 5 ’and 3’ ends of these mRNA.Recent studies have shown that transcription factors(TFs)and miRNAs work together to control the development of B cells at many checkpoints.In order to further explore the role of miRNAs in the development and activation of B cells,microarray was conducted and showed that the expression of miR-130b in different immune cells was different.In addition,q-PCR detection suggested that the expression of miR-130b was lowest in ProPre B cells and highest in immature B at different stages of B cell development.However,the expression of miR-130b was decreased in mature B cell,and further decreased after activation.These results suggested that miR-130b may be related to B cell development and activation.Therefore,in this study,we propose to further study the role of miR-130b in B cell activation by establishing the model of miR-130b overexpression bone marrow chimeric mice,miR-130b knockout mice and miR-130b transgenic mice.First of all,we infected bone marrow stem cells with miR-130b retrovirus,and established the model of miR-130b overexpression bone marrow chimeric mice.FACS analysis and q-PCR proved that our miR-130b-overexpressed bone marrow chimeric mice model was successfully established.Meanwhile,we established miR-130b knockout mice and miR-130b transgenic mice models.Through the immuno-phenotypic analysis of miR-130b bone marrow chimeric,knockout and transgenic mice,we found that miR-130b mainly affected the proliferation and activation of B cells and B cell apoptosis.CD80 and CD86 on the surface of B cells were significantly increased in bone marrow chimeric mice,but the difference was not found in transgenic mice.This may be due to the different degree of over expression of miR-130b in the bone marrow chimeric mice and transgenic mice.At the same time,when using LPS,or anti-IgM antibody,or anti-IgM plus anti-CD40 antibody to activate the naive B cells,we can find that the proliferation and activation of miR-130b overexpressed B cells is significantly higher than that of the control group,on the contrary,the proliferation of miR-130b knockout B cells is decreased compared with that of WT B cells.Calcium influx is an important event in B cell activation,flow cytometry analysis showed that the release of intracellular calcium in B cell of miR-130b transgenic mice was higher than that of the control group,and the expression of NFAT1 was significantly increased.Annexin V detection showed that overexpression of miR-130b could inhibit B cell apoptosis.The mRNA and protein levels of Bcl-2 in miR-130b overexpressed B cells were significantly increased,suggesting that miR-130b may inhibit B cell apoptosis by affecting the expression of Bcl-2.Softwares analysis,q-PCR and luciferase reporting assay together suggest that Cyld could be the target gene of miR-130b in B cells,The expression of Cyld was decresed in miR-130b over-expressed B cells,while the expression of TRAF2 and NF-κB were increased,and the phosphorylated NF-κB was also increased significantly.Therefore,we successfully established the animal models of miR-130b bone marrow chimeric mice,knockout mice and transgenic mice,which provided a powerful research tool for further study of the biological function of miR-130b.our existing studies suggest that miR-130b can affect NF-κB activity,promote B cell activation and inhibit B cell apoptosis by targeting Cyld.It provides a new theoretical basis for the molecular regulation of B cell activation.