Effect Of Lipopolysaccharide On MicroRNA-107 And NF-kB Signaling Pathways In U937 Cells

Objective:To investigate the relationship between microRNA-107 and NF-kB signaling pathways in monocytes under inflammatory conditions,U937 cells were stimulated with lipopolysaccharide(LPS)to construct an inflammation model.And explore the mechanism of action of both in promoting the inflammatory state of patients with diabetic neuropathy.Methods:1.In vitro application of Phorbol 12-myristate 13-acetate(PMA)induced the maturation of human macrophage U937 cells.Then U937 cells were stimulated with different concentrations of LPS(0.05-10 ?g/ml)and cells were harvested 3 hours later.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the gene mutation of tumor necrosis factor-?(TNF-?),and the optimal concentration of LPS was also selected2.In accordance with the LPS stimulation time(10 minutes,1 hour,3 hours,6 hours)were divided into 4 groups,each time point set the control group and LPS(10?g/ml)stimulation group.Enzyme-linked immunosorbent assay(ELISA)and qRT-PCR were used to detect the expression of inflammatory cytokines TNF-? and Interleukin(IL-1?).3.In accordance with the LPS stimulation time(10 minutes,1 hour,3 hours,6 hours)were divided into 4 groups,each time point set the control group and LPS(10?g/ml)stimulation group.Polyacrylamide gel electrophoresis(Western blot,WB)was used to detect the protein levels of TLR4,p65 and phospho-p65(Ser536).4.Effects of microRNA-107 Inhibitors on U937 Cell Inflammatory Responses Grouping:control group;inhibitor group;LPS group;LPS+inhibitor group.ELISA and RT-PCR were used to detect the expression of TNF-? and IL-1?Results:1.After U937 cells were induced by PMA for 24 hours,U937 cells adhered to the wall and protruded pseudopodia.LPS was then added to construct the inflammation model.The concentration of LPS was 10 ?g/ml.2.Compared with untreated control group,LPS stimulated the expression of microRNA-107,TNF-? and IL-1? mRNA in all groups significantly(p<0.05),and the expression levels of TNF-? and IL-1? increased significantly(p<0.05).Spearman correlation analysis showed that microRNA-107 was positively correlated with NF-?B downstream target gene TNF-? mRNA(r=0.773,p<0.01),IL-1?mRNA(r=0.801,p<0.01)was positively correlated.3.The expression of p-NF?B p65(Ser536)/p65 in U937 cells at 1 hour,3 hours and 6 hours was higher than that in the control group(p<0.01).The Spearman correlation analysis showed that the expression of microRNA-107 in U937 cells stimulated by LPS was positively correlated with the expression of p-p65(Ser536)/p65 protein(r=0.656,p<0.01).4.Compared with the control group,the expression of microRNA-107,TNF-?and IL-1? in LPS group increased(p<0.01);compared with LPS group,the expression of microRNA-107,TNF-? and IL-1? in LPS+inhibitor group The amount was significantly decreased(p<0.01)Conclusion:The increase of microRNA-107 expression induced by lipopolysaccharide in phorbol ester-induced U937 cells may be related to NF-?B(p65)signaling pathway.MicroRNA-107 inhibitor can reduce the secretion of inflammatory factors.