Effect Of MiR-155 On The Secretion Of Th2 Type Cytokines By Mouse ILC2

Objective:The spleen ILC2 cells were sorted in vitro to investigate the effect of miR-155 on the secretion of Th2 inflammatory cytokines by ILC2 and its mechanism.Methods:1.After 6-8 weeks of wild-type BALB/c female mice were sacrificed by cervical dislocation,the spleens of the mice were removed from the aseptic processing table,and then the lymphocyte separation solution was added for grinding,and then centrifuged in a centrifuge,then the lymphocyte layer is aspirated to obtain spleen mononuclear cells.2.Wild-type BALB/c mouse spleen mononuclear cells were sorted by immunomagnetic beads enrichment and flow cytometry for primary culture of ILC2,and cytokines IL-33 and IL-7 were added for expansion and culture for about ten days,the microscope observes whether there is proliferation.3.After the expanded cells were removed from the cell culture medium,new medium was added and resuspended in the culture flask,and then the cells were randomly divided into four groups.Each group of cells was transfected with miR-155 agomir,agomir NC(miR-155 agomir control),miR-155 antagomir and antagomir NC(miR-155 agomir control)for subsequent experiments.qPCR was used to detect the mRNA expression of miR-155 and c-maf in each group of cells.Western Blot was used to detect the expression of c-maf protein in each group of cells.The expression levels of IL-4,IL-5,IL-9 and IL-13 in the supernatant of each group were detected by ELISA.Results:1.In the transfected miR-155 agomir group,the expression of miR-155 was significantly increased(P < 0.001),whereas the expression of mir-155 was decreasedin the transfected miR-155 antagomir group(P < 0.01).It was demonstrated that miR-155 has been transfected into the target cells.2.The mRNA expression of c-maf was decreased in the miR-155 agomir group(P<0.01),while the miR-155 antagomir group was increased(P<0.01).The expression of c-maf protein in miR-155 agomir group was lower than that in the control group by Western Blot(P<0.001),while the miR-155 antagomir group was higher than the control group(P<0.05).The difference was statistically significant.3.ELSIA detected that IL-4,IL-9 and IL-13 were elevated in miR-155 agomir group,but IL-4 and IL-13 in miR-155 antagomir group were decreased;however,there was no change in the expression level of IL-5 in the up-regulated group or the down-regulated group.Conclusion:1.miR-155 can promote the secretion of Th2 type cytokines such as IL-4,IL-9and IL-13 by mouse ILC2.2.miR-155 can affect the expression of c-maf protein in ILC2,which may be an important cause of the secretion of Th2 type cytokines by ILC2.