The Role Of HSP70 And NF-?B In TNF-? Induced Apoptosis Of A549 Cells

Background and objectiveLung cancer is one of the malignant tumors with high incidence rate and high mortality rate worldwide,which seriously endangers people's life and health.A large number of studies have shown that lung cancer cells can antagonize the regulation of cell proliferation and apoptosis through various ways in the body,resulting in the malignant proliferation of lung cancer cells in the body tissue,thus damaging the normal tissue and cell functions in the body.Recent studies have confirmed that Tumor necrosis factor-?(TNF-?)can induce the apoptosis of Tumor cells with a wide range of specific anti-tumor effects,and as a complex bioactive cytokine secreted by immune cells,it can mediate a variety of cellular responses including proliferation,differentiation,inflammation and apoptosis.The Nuclear transcription factor-?B(NF-?B)is the signal transduction core of TNF-?-induced inflammatory response,and can bind to a variety of enhancers and promoters to inhibit the activity of NF-?B,which can theoretically block the changes in TNF-?-activated inflammatory response,cell apoptosis and oxidative stress.Heat shock protein-70(HSP70)belongs to the family of heat shock proteins(HSPs)as one of the most important member,HSP70 can be used as a molecular chaperone involved in protein folding,assembly,and degradation of biological processes,some studies have found that overexpression of HSP70 in lung cancer tissues is associated with poor prognosis of lung cancer patients,and high expression of HSP70 is often beneficial to the survival of tumor cells.In previous experiments,TNF-? induced apoptosis process in A549 cells accompanied by increased levels of Non-phagocytic cell oxidase-1(NOX1)protein and reactive oxygen species(ROS),as an important member of the nicotinamide adenine dinucleotide phosphate(NADPH)oxidase family,the ROS produced by NOX1 is essential to maintain the redox balance in the body.Therefore,the purpose of this study is to explore whether HSP70 has an effect on the apoptosis process induced by TNF-? and its possible mechanism,at the same time,the role of NOX1 protein and NF-?B signaling protein in the apoptosis of A549 cells induced by TNF-? was verified,and the relationship among HSP70,NOX1 and NF-?B was investigated.Therefore,it provides theoretical basis and experimental basis for the validation of HSP70 protein as a potential target in the diagnosis and treatment of lung cancer.Methods1.Experimental group:(1)When the apoptosis model was constructed,it was divided into 0.00nmol/L group,0.25nmol/L group,0.50nmol/L group,1.00nmol/L group according to TNF-? concentration;(2)When inhibiting the expression of HSP70,they were divided into blank group,TNF-? group,VER155008(HSP70 inhibitor)group and TNF-?+VER155008 group;(3)When inhibiting the expression of NF-?B,they were divided into blank group,TNF-? group,BAY11-7082(NF-?B inhibitor)group and TNF-?+BAY11-7082 group.2.Observation of cell morphology:Living cell workstation was used to take pictures of cells in each group and observe the changes of cell morphology among groups.3.Cell proliferation activity assay:CCK-8 test kit combined with enzyme labeling instrument was used to detect the proliferation activity of each group of cells,and the proliferation activity of cells in each group was determined according to the absorbance of cells in each group.4.Protein detection:The expression of HSP70?NOX1 protein and signal transduction proteins NF-?B was detected by Western Blot.5.Apoptosis detection:AnnexinV-FITC/PI apoptosis assay kit was used in combination with flow cytometry to detect the apoptosis of each cell group.6.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 four 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 a=0.05.Results1.When the TNF-? concentration was 0.50nmol/L,TNF-? stimulated A549 cells for 24 hours can significantly reduce cell proliferation activity and promote cell apoptosis,accompanied by increased expression of NOX1 protein and NF-?B signaling protein(P<0.05),but has no effect on HSP70 protein expression(P>0.05);Therefore,the 0.50nmol/L TNF-?was selected for follow-up experiments.2.TNF-?+VER1 55008 group compared with TNF-? group,HSP70,NOX1 proteins and NF-?B signaling proteins were significantly decreased(P<0.05);VER155008 group compared with blank group,HSP70,NOX1 proteins and NF-?B signaling proteins were significantly decreased(P<0.05);The results of CCK-8 experiment showed that the proliferation activity of cells in TNF-?+VER1 55008 group was further decreased compared with that in TNF-? group(P<0.05);The results of flow cytometry showed that compared with the TNF-? group,the apoptosis rate of the TNF-?+VER155008 group was further increased(P<0.05).These results suggested that inhibition of HSP70 expression could promote TNF-? induced apoptosis of A549 cells.3.TNF-?+BAY1 1-7082 group compared with TNF-? group,NOX1 proteins and NF-?B signaling proteins were significantly decreased(P<0.05),but has no effect on HSP70 protein expression(P>0.05);BAY11-7082 group compared with blank group,NOX1 proteins and NF-?B signaling proteins were significantly decreased(P<0.05);The results of CCK-8 experiment showed that the proliferation activity of cells in TNF-?+BAY11-7082 group was increased compared with that in TNF-? group(P<0.05);The results of flow cytometry showed that compared with the TNF-? group,the apoptosis rate of the TNF-?+BAY11-7082 group was decreased(P<0.05).These results suggested that NOX1 and NF-?B signaling proteins play an important role in the apoptosis of A549 cells induced by TNF-?.ConclusionsThe results of this study suggest that NOX1 and NF-?B proteins can mediate the apoptosis of A549 cells induced by TNF-?;Decreasing HSP70 expression in A549 cells can promote TNF-? induced apoptosis through NF-?B/NOX1 signaling pathway,and HSP70 may be a potential target for the treatment of lung cancer.