Role Of NADPH Oxidase 4 In Epithelial-Mesenchymal Transition Of A549 Cells

Background and objectiveLung cancer is a malignant tumor that seriously affects human health.One of the main reasons of death from lung cancer patients is the metastasis of lung cancer.The epithelial-mesenchymal transition(EMT)process is one of the important mechanisms of lung cancer metastasis,and it is also an important step to promote tumor metastasis.Therefore,exploring the mechanism of cell EMT process will provide a theoretical basis for the treatment of lung cancer metastasis.Transforming growth factor-?(TGF-?)is a kind of multi-functional cytokine,which is the classical inducer of cellular EMT model in vitro.Nicotinamide adenine dinucleotide phosphate 4(NOX4)is an important type of NOX family and its main role is to produce reactive oxygen species(ROS).ROS can be used as a second messenger to promote the transduction of some signaling pathways in cells,thereby regulating the biological behaviors of cells such as proliferation,apoptosis,differentiation,and migration.MAPK(Mitogen-activated protein kinase)signaling pathway mediates TGF-?-induced cellular EMT process in breast cancer cells.TGF-? can activate the NF-?B(Nuclear factor-?B)signaling pathway,which in turn promotes the EMT process in human malignant glioblastoma cells.In previous experiments,TGF-? induced EMT process in A549 cells accompanied by increased levels of NOX4 protein and ROS,and promoted cell migration.So,whether MAPK and NF-?B signaling pathway mediate TGF-?-induced EMT process in A549 cells? What is the relationship between MAPK,NF-?B and NOX4 in the EMT process of A549 cells? In order to explore the above issues,The A549 cells were stimulated with TGF-? to construct EMT model.Then,the DPI,SB203580,and BAY11-7082 were used to inhibit NOX4,p38 MAPK,and NF-?B(p65)respectively.Finally,the effects of these inhibitors on cell EMT process,proliferation activity and cell migration ability were observed.Methods1.Experimental group:(1)When the EMT model was constructed,it was divided into 0.0 ?g/L group,1.0 ?g/L group,2.5 ?g/L group,5.0 ?g/L group,and 10.0 ?g/L group according to TGF-? concentration;(2)When inhibiting the expression of NOX4,they were divided into blank group,TGF-? group,TGF-?+DPI group,DPI(NOX4 inhibitor)group and DMSO(solvent control)group;(3)When p38 MAPK expression was inhibited,they were divided into: blank group,TGF-? group,TGF-?+SB203580 group,SB203580(p38MAPK inhibitor)group,and DMSO(solvent control)group;(4)When the expression of NF-?B was inhibited,it was divided into blank group,TGF-? group,TGF-?+BAY11-7082 group,BAY11-7082(NF-?B inhibitor)group,and DMSO(solvent control)group.2.Protein detection: Western Blot was used to detect the expression of NOX4 protein,EMT-related proteins E-cadherin,Vimentin,Snail,and signal transduction proteins p38 MAPK,p-p38 MAPK,JNK,p-JNK,NF-?B(p65),and p-NF-?B(p-p65).3.mRNA level detection: NOX4 mRNA,p38 MAPK mRNA,and NF-?B(p65)mRNA levels were detected by Real-Time PCR.4.Reactive oxygen detection : The level of reactive oxygen species was qualitatively detected by a laser confocal microscope and the level of reactive oxygen species was quantitatively measured by flow cytometry.5.Observation of cell morphology: The cells were photographed by a living cell station and their cell morphology was observed.6.Migration ability test: The cell migration ability was determined by scratch test,and the scratch width of the cells was analyzed using photoshop software,and the cell migration ability was judged based on the scratch width.7.Cell proliferation activity assay: The MTT cell proliferation activity detection kit was combined with a microplate reader to detect the proliferation activity of the cells,and the proliferation activity of the cells was judged based on the absorbance of different groups.8.Statistical analysis: The raw data obtained by the experiment were expressed as the mean plus or minus standard deviation(?x±s),and the data was analyzed statistically using SPSS 21.0 software.The one-way analysis of variance(one-way ANOVA)was used to compare the means of the five component groups.The least significant difference method(LSD-t test)was used to compare the mean of each group of data.The mean of the two component groups were compared using two independent samples t-test.Test level ? = 0.05.Results1.When the TGF-? concentration was 5.0 ?g/L,the epithelial cell marker E-cadherin protein was significantly reduced and the interstitial cell marker Vimentin protein and EMT process transcription factor Snail were significantly elevated,and the expression of NOX4 protein and the level of ROS was increased(P<0.001).Therefore,the 5.0 ?g/L TGF-? was selected for follow-up experiments.2.The expression of p38 MAPK,p-p38 MAPK,NF-?B(p65),and p-NF-?B(p-p65)was increased compared with the control group after stimulated by TGF-? in A549 cells(P<0.05).3.TGF-?+DPI group compared with TGF-? group,the NOX4 mRNA level,NOX4,p38 MAPK,p-p38 MAPK,NF-?B(p65),p-NF-?B(p-p65),Vimentin,Snail protein expression and level of ROS were decreased,but the E-cadherin protein was increased(P<0.001).MTT results showed that the proliferation activity of TGF-?+DPI group was lower than that of TGF-? group(P<0.001).The scratch test showed that the width of the scratches in the TGF-?+DPI group was wider than that in the TGF-? group(P<0.001),indicating that the potential ability was reduced..4.TGF-?+SB203580 group compared with TGF-? group,the p38 MAPK mRNA level,p38 MAPK,p-p38 MAPK,NOX4,Vimentin,Snail protein expression and the level of ROS were decreased,but the E-cadherin protein was increased(P<0.001).Meanwhile,the proliferative activity and cell migration were both decreased(P<0.001).5.TGF-?+BAY11-7082 group compared with TGF-? group,the NF-?B(p65)mRNA level,NF-?B(p65),p-NF-?B(p-p65),NOX4,Vimentin,Snail protein expression and the level of ROS were decreased,but the E-cadherin protein was increased(P<0.001).The proliferative activity and cell migration were both decreased(P<0.001).Conclusions1.NOX4,p38 MAPK and NF-?B(p65)can mediate the process of EMT in A549 cells.NOX4 and p38 MAPK,NOX4 and NF-?B(p65)interact with each other in the process of EMT induced by TGF-? in A549 cells.And they promote EMT process in A549 cells together.2.Inhibition of NOX4,p38 MAPK,and NF-?B(p65)by DPI,SB203580,and BAY11-7082 respectively can reverse TGF-?-induced EMT process,and reduced cell proliferative activity and cell migration finally in A549 cells.