PM2.5 Exposure Induced Sustained Activation Of NF-?B In Airway Epithelial Cells Via MiR-331/IKK-? Axis

PM2.5(fine particulate matter)refers to fine particulate matter with aerodynamic diameter less than or equal to 2.5?m,which is seriously harmful to social environment and human health.A large number of studies have shown that a variety of respiratory diseases in humans have a significant correlation with PM2.5,but the mechanism of PM2.5 on respiratory system-related diseases is unclear.Therefore,we choose human bronchial epithelial cells(BEAS-2B Cell,B2B)to investigate the effects of PM2.5 on the biological function of B2 B cells and the related molecular signaling pathways involved in the regulation.In this study,we used PM2.5(25 ?g/cm 2)and LPS(5 ?g/ml)to stimulate the B2 B cells for 24 h and observed the expression of NF-?Bp65 at different time points.Immunofluorescence staining and Western blot showed that both PM2.5 and LPS could increase the expression of NF-?Bp65 in the nucleus.NF-?Bp65 was rapidly activated after 1 h of LPS stimulation and returned to the original state after 6 h of LPS stimulation,while NF-?Bp65 was continuous activated after 6 h of PM2.5exposure,and continued to 24 h,suggesting that it was different between PM2.5 and LPS about NF-?B inflammatory signal activation,PM2.5 can promote NF-?Bp65sustained activation.In order to study the molecular mechanism of PM2.5 on NF-?Bp65 regulation,the expression of miR-331 in PM2.5-exposed B2 B cells was detected by qRT-PCR.Results showed that miR-331 expression was significantly down-regulated(P<0.01)after 12 h and 24 h of PM2.5 exposure.B2 B cells pretreated with Ly294002(PI3K kinase selective inhibitor)and NAC(ROS scavenger)were exposed to PM2.5 for 12 h and 24 h,the results showed the expression of miR-331 in the B2 B cells was Increased compared with those without pre-treated B2 B cells(P<0.05),suggesting that PM2.5 downregulated the expression of miR-331 through ROS/PI3K/Akt signaling pathway in B2 B cells.In addition,the cells were transfected with miR-331 mimics and the expression of NF-?Bp65,IL-6 and IL-8 were observed after exposure to PM2.5 for24 h.Immunofluorescence staining,Western blot,ELISA andqRT-PCR showed that the expression of NF-?Bp65,IL-6 and IL-8 were decreased(P<0.05),indicating that miR-331 Weaken the inflammatory response of B2 B cell by PM2.5.To further explain how miR-331 is involved in the PM2.5-induced inflammatory response mechanism,we searched for the potential target of miR-331 through the TargetScan database and found that the 3'UTR of the IKK-?(NF-?B inhibitor kinase)has miR-331 binding site.The luciferase reporter assay confirmed that miR-331 binds to this site to regulate luciferase expression.Western blot and qRT-PCR showed that the expression of IKK-? was significantly down-regulated by miR-331 mimics compared with the control group(P<0.01).The above results indicated that miR-331 could negatively regulate the expression of IKK-? by targeting IKK-?3'UTR.To determine whether IKK-? is involved in miR-331-induced inflammatory response to PM2.5,we constructed B2 B cells overexpressing IKK-? while transfected with miR-331 mimics and exposed to PM2.5.The results showed that overexpression of IKK-? could block the expression of on PM2,IL-6 and IL-8 in promoting by miR-331 in B2 B cells(P<0.01).In conclusion,we conclude that PM2.5 exposure reduces the expression of miR-331 in human bronchial epithelial cells through the ROS/PI3K/Akt signaling pathway,as miR-331 negatively regulate the expression of IKK-? by targeting IKK-3'UTR,Therefore,an increasing expression of IKK-? promote the activation of NF-?B and releasing of related inflammatory factors.In short our results provided new reference new information for the pathogenesis of respiratory system exposure to PM2.5.