Effects Of MicroRNA-140-5p On Phenotype And Function Of Septic Dendritic Cells

Objective:To observe the effect of sepsis on the expression of surface molecules and apoptosis of DC,and to detect the changes of miR-140-5p expression in sepsis DC.Methods:?Mononuclear cells were extracted from human peripheral blood,and monocytederived dendritic cells(monocyte-derived dendritic cells,MoDC)were cultured in vitro induced by recombinant human cytokine interleukin-4(IL-4)and granulocytemacrophage colony stimulating factor(GM-CSF).?Experimental grouping and cell treatment methods:(1)NC group: On the fifth day of MoDC culture in vitro,LPS with 1ug/ml was added for 48 hours;(2)sepsis model group: Continuous stimulation was given by LPS of 1ug/ml in the whole process of culture;(3)-(6)sepsis + miRNA-transfected group: same as sepsis model group,transfection(miR-140-5pmimics,inhibitor and corresponding control)on the fifth day of culture for 12 hours and then treated with 1ug/ml concentration of LPS for 48 hours.? Flow cytometry was used to detect the expression of DC surface molecules(CD80,CD86 and HLA-DR)and apoptosis in different treatment groups.? The intracellular fluorescence signal after transfection was detected by fluorescence microscope and the expression of miR-140-5p was detected by q-PCR to verify the transfection effect.? Enzyme labeling instrument to detect the effect of DC on T cell proliferation in different treatment groups,q-PCR to detect the expression of TLR-4 and NF-? B mRNA,and Western blot to detect the expression of TLR-4,NF-? B and Bcl-2.Results:? Compared with the control group treated with LPS short-term stimulation,the expression of miR-140-5p and the expression of CD80,CD86 and HLA-DR on dendritic cells of septic model in vitro after continuous LPS stimulation decreased significantly,while apoptosis increased significantly(p < 0.01),and the effect of stimulating T cell proliferation was weakened(p < 0.0l).? Transfection of mimics and inhibitor could well interfere with the expression of miR-140-5P,overexpression of miR-140-5p could improve the down-regulated expression of CD80,CD86 and HLA-DR on septic DC(p<0.05),decrease the apoptosis of dendritic cells(p<0.01)and enhance the effect of stimulating T cell proliferation(p<0.05).? The expressions of TLR-4 and NF-? B(p65)mRNA and protein were upregulated in septic dendritic cells(p<0.05),and overexpression of miR-140-5p could inhibit it's expression(p<0.05).The expression of bcl-2 in septic dendritic cells was inhibited(p<0.05),and overexpression of miR-140-5p could improve the inhibition(p<0.05).Conclusion:In sepsis,the expression of miR-140-5P in dendritic cells was decreased,the maturation of dendritic cells was inhibited,the apoptosis of dendritic cells was increased,and the effect of stimulating T cell proliferation was weakened.Overexpression of miR-140-5p can improve the inhibition of DC maturation in sepsis,reduce the apoptosis of dendritic cells and enhance the effect of stimulating T cell proliferation.The mechanism may be that targeted TLR-4 affects the phenotype and function of DC through TLR-4/NF-? b signal pathway and regulation of apoptosisrelated protein bcl-2.The purpose of this study is to provide experimental basis for the early treatment strategy of sepsis through regulating DC function by exogenous miRNA to reverse the immunosuppression of sepsis.