The Role Of TNF-α In TGF-β-induced EMT Process In A549 Cells

Background and ObjectiveLung cancer is one of the most common malignant tumors worldwidealthough breakthroughs have been made in treatment methods in recent years such as targeted therapy and immunotherapy,and relatively satisfactory results have been achieved,lung cancer still has the highest morbidity and mortality in the world.About 1.6 million people die from lung cancer each year,the overall 5-year survival rate is only 16%,posing a serious threat to human health and life.Studies have shown that the proliferation and metastasis of cancer cells are the main factors leading to poor prognosis of lung cancer patients.About 90%of the deaths of cancer patients are due to tumor invasion and metastasis,and epithelial-mesenchymal transition(EMT)plays an important role in this process.When lung tissue epithelial cells are carcinogenically stimulated by dust,bleomycin,smoking and other factors,they may become cancerous.The process of canceration is accompanied by the occurrence of the EMT process,which causes the cells to lose the characteristics of epithelial cells,showing more mesenchymal cell characteristics,enhanced migration and invasion potential.Transforming growth factor-β(TGF-β)plays an important biological function in inflammation,embryonic development,epithelial-mesenchymal transition(EMT),tissue repair and migration.In the tumor microenvironment,TGF-β induces the transition of lung epithelial cells to mesenchymal cells by controlling the deposition of extracellular matrix,thereby affecting the changes of tissue structure.Therefore,exploring the mechanism of inhibiting tumor cell epithelial-mesenchymal transition has become a research hotspot.Recently,a large number of studies have shown that Tumor necrosis factor alpha(TNF-α)is exists widely in tumor cells and plays a very important role in the process of EMT.As an important inflammatory regulator,TNF-α plays an important role in cell homeostasis,immune regulation,inflammation and apoptosis.Nuclear factor-κB(NF-κB)is a group of transcription factors widely present in mammalian cells.In addition to participating in autoimmune response and inflammation,it also participates in the occurrence of skin appendages and bone remodeling.A large number of studies have found that the NF-κB signal pathway can regulate important physiological functions such as cell proliferation,differentiation,and apoptosis,and is closely related to tumor occurrence,development,metastasis,and tumor cell apoptosis.Non-phagocytic oxidase 4(NOX4)is an important member of the nicotinamide adenine dinucleotide phosphate(NADPH)oxidase family,and its main function is to generate reactive oxygen species(ROS),maintain the redox balance in the body.The ROS produced by it can mediate cell proliferation,migration and differentiation,and it has been proven to promote the EMT process.So,does TNF-α affect the EMT process of lung cancer cells?Does TNF-αpromote or inhibit the EMT process of lung adenocarcinoma cells represented by A549 cells?Is there a relationship between TNF-α and NF-κB,NOX4,and ROS in these processes?Therefore,this experiment uses TGF-β to stimulate lung tissue epithelial cells A549 cells to construct an EMT model,and then uses TNF-α to act on each group of cells to explore the effect of TNF-α on the EMT model,at the same time discusses the role and relationship of TNF-α、NF-κB signaling proteins and NOX4 protein in the process.Therefore,it provides corresponding experimental and theoretical basis for the prevention,diagnosis and treatment of lung cancer.Methods1.Construction of the EMT model of A549 cells:When A549 cells adhered to the wall and covered about 80%of the bottom of the cell bottle,they were stimulated with 5 μg/L TGF-β for 24 h to establish the EMT model.2.Experimental group:(1)Screening of TNF-α concentration:it was divided into 0.00 μg/L,0.01 μg/L,0.02 μg/L,0.03 μg/L,0.04 μg/L and 0.05 μg/L group according to TNF-α concentration.CCK-8 was used to detect the proliferation activity of A549 cells to determine the optimal concentration,and the optimal concentration of TNF-α was selected for subsequent experiments.(2)The effect of TNF-α on EMT process was investigated and divided into control group,TGF-β group,TNF-α group and TGF-β+TNF-α group.(3)When explore the effect of TNF-α on NF-κB signaling pathway and NADPH oxidase,they were divided into control group,TGF-β group,TNF-α group and TGF-β+TNF-αgroup.(4)When inhibiting the expression of NF-κB,they were divided into contorl group,TGF-β group,TGF-β+TNF-α group,TGF-β+BAY11-7082(NF-κB inhibitor)group,TGF-β+TNF-α+BAYl 1-7082 group.3.Detection of cell proliferation activity:CCK-8 detection kit and enzyme labeling instrument were used to detect cell activity of each group,and calculate and compare cell proliferation activity by detecting the absorbance of each group of cells.4.Observation of cell morphology:observe each group of cells through the live cell workstation and representative cells were selected to take pictures,the cell morphology of each group was observed and compared for analysis.5.Detection of cell migration ability:the cell migration ability was detected by scratch experiment,and the scratches were analyzed by using Photoshop software,which was represented by relative width.6.Detection of protein levels:the expression of EMT marker protein E-cadherin,Vimentin,NOX4 protein and signaling pathway protein NF-κB(p65)protein were detected by Western Blot.7.Statistical analysis:The original data obtained in the experiment are expressed in the form of mean±standard deviation(xts,n=3),and all the data obtained in the experiment was analyzed by SPSS27.0 statistical software.The one-way analysis of variance(one-way ANOVA)was used to compare the means of multiple groups.The least significant difference method(LSD-t test)was used to compare the mean of each group of data.Two independent sample t-test is used to determine the grouping of only two groups of mean comparison,set detection level of a=0.05.Results1 When the concentration of TNF-α was 0.02 μg/L,stimulate A549 cells for 24 h,and it was found that compared with 0.00 μg/L group,the cell proliferation activity is reduced(P<0.001).Select 5 μg/L TGF-β to stimulate A549 cells for 24 h.Compared with control group,the expression of E-cadherin protein in TGF-β group was significantly reduced,while the expression of Vimentin protein was obviously increased,and the scratch experiment showed that the width of the scratch between cells decrease(P<0.001),indicating that its migration ability is enhanced.2.Compared with TGF-β group,the expression of E-cadherin protein in TNF-α+TGF-β group was significantly increased,while the expression of Vimentin protein was decreased(P<0.001);the scratch test showed that the width of the scratches between cells in the TNF-α+TGF-β group was wider than that in TGF-β group(P<0.001),indicating that the cell migration ability is decreased.3.Compared with control group,the expression of NF-κB and NOX4 protein in TGF-β group increased(P<0.001);compared with TGF-P group,the expression of NF-κB and NOX4 protein in TNF-α+TGF-β group was significant decreased(P<0.001).4.Compared with control group,the cells in TGF-β group showed a long spindle shape and the intercellular space was enlarged;compared with TGF-β group,the cells in TGF-β+BAY11-7082 group had blunt edges and fewer cells.The TGF-β+TNF-α+BAY11-7082 group was more obvious,most of the cells showed the blunt round shape of epidermal cells.Compared with TGF-β group,the expression of E-cadherin protein in TGF-β+BAY11-7082 group was significantly increased,while the expression of Vimentin,NF-κB(p65)and NOX4 protein was decreased,and the difference was statistically significant(P<0.001).Compared with TGF-(3+TNF-α group,the expression of Vimentin protein in TGF-β+TNF-α+BAY11-7082 group was significantly increased,while the expression of E-cadherin,NF-κB(p65)and NOX4 protein were all decreased(P<0.001).ConclusionsTNF-α inhibited the EMT process of A549 cells by inhibiting the NF-κB/NOX4 signaling pathway activated by TGF-β stimulation,and ultimately reduced the migration ability of A549 cells,thus reducing the proliferation and metastasis of lung cancer cells.