| Background and objective: Epithelial mesenchymal transition(EMT)is the process by which epithelial cells lose their polarity and acquire the peculiarity of mesenchymal cells.EMT plays an important role in embryonic development,tissue repair and fibrosis.The main reason of epithelial-mesenchymal transition is that the epithelial cells of lung tissue are stimulated,especially after the stimulation of transforming growth factor-β(TGF-β),the epithelial cells are abnormally repaired and transited into mesenchymal cells.During the transition from epithelial cells to mesenchymal cells,the intermediate cell morphology-myofibroblasts is formed,secreting extracellular matrix proteins(ECM),resulting in excessive deposition,which eventually leads to pulmonary dysfunction.TGF-β is a multifunctional cytokine and a classic cytokine for the construction of EMT models in vitro.When cells are stimulated by ischemia,high heat,toxins,viral particles,etc.a group of highly conserved proteins is synthesized,known as "heat shock proteins"(HSPs).Recent studies have shown that the drug of geranylgeranylacetone(GGA),which is clinically used to treat gastric ulcers,has been reported as a non-toxic inducer of HSP70.Therefore,this study established an EMT model in vitro by stimulating A549 cells with TGF-β;then,high expression of HSP70 was induced by GGA to explore the effect of HSP70 on EMT model.In this experiment,and the expressions of NOX4,HSP70 and EMT-related proteins E-cadherin and vimentin were detected by western blot,the morphological changes of cells were observed by the living cell workstation,and the changes of cell migration ability of HSP70 were detected by scratch assay,so as to explore the regulatory effect of HSP70 on EMT process.Methods: 1.TGF-β stimulates A549 cells to construct EMT model The experiment was divided into the blank group and the model group.The blank group was only added with RPMI1640 medium,and the model group was added TGF-β with a final concentration of 5μg/L.After A549 cells were stimulated for 24 h, proteins in each group were collected,and the expressions of E-cadherin,vimentin,HSP70 and NOX4 proteins in each group were detected by western blot.2.Selecting the optimal concentration and optimal time of GGA.Firstly,A549 cells were stimulated with GGA of different concentrations for 24 hours,after which,proteins of each group were collected.Then,the expression of HSP70 was detected by western blot to select the optimal concentration.According to the optimal concentration,A549 cells were stimulated at different time points(0h,1h,2h,4h,8h,12 h and 24h)respectively,after which,proteins at different time points were collected,then HSP70 expression was detected by western blot to select the best time.3.Effect of HSP70 induced by GGA on EMT of A549 cells According to the selected optimal GGA concentration,the experiment is divided into 50μmol/L and 200μmol/L;each concentration was divided into 5 groups,namely,blank group,DMSO group,TGF-β group,TGF-β+GGA group,GGA group.Proteins in each group were collected and the expressions of EMT-related proteins E-cadherin and vimentin in each group were detected by western blot.4.Effect of HSP70 induced by GGA on the morphology of A549 cells According to the selected optimal GGA concentration,the experiment is 50μmol/L and 200μmol/L;each concentration was divided into 5 groups,namely,blank group,DMSO group,TGF-βgroup,TGF-β+GGA group,GGA group.The living cell workstation was used to observe the morphological changes of cells and take pictures of them.5.Effect of HSP70 induced by GGA on the migration ability of A549 cells According to the selected optimal GGA concentration,the experiment is divided into 50μmol/L and 200μmol/L;each concentration was divided into 5 groups,namely,blank group,DMSO group,TGF-β group,TGF-β+GGA group,GGA group.Scratch assay was applied to detect GGA-induced HSP70’s changes in A549 cell migration ability.Results 1.Compared with the blank group,the expression level of E-cadherin was decreased in EMT model group of the A549 cell induced by TGF-β,the expression levels of HSP70,vimentin,NOX4 and in the EMT model group were increased,and the difference was statistically significant(P<0.05).2.When selecting the optimal GGA concentration of stimulating A549 cells,compared with the 0μmol/L group,the HSP70 protein expression level of the 50μmol/L group was increased;compared with the 100μmol/L group,the expression level of HSP70 protein in the 200μmol/L group increased,and the difference was statistically significant(P<0.05).When selecting the optimal time for GGA to stimulate A549 cells,the expression level of HSP70 protein increased at 24 h,and the difference was statistically significant(P<0.05).3.When A549 cells were treated with 50μmol/L GGA,the effect of HSP70 on EMT-related proteins was observed: compared with the blank group,the expression of E-cadherin is decreased,the expression of vimentin increased in the TGF-β group;the expression of E-cadherin and vimentin was increased in the TGF-β+GGA group;the expression of vimentin increased in GGA group;compared with the TGF-β group,the expression level of vimentin in the TGF-β+GGA group increased,while the expression level of E-cadherin in the GGA group increased;compared with the TGF-β+GGA group,the expression level of E-cadherin increased,while the expression level of vimentin decreased in GGA group,and the difference was all statistically significant(P<0.05).When A549 cells were treated with 200mol/L GGA,the effect of HSP70 induced by it on EMT-related proteins was observed: compared with the blank group,the expression level of E-cadherin was decreased,the expression of vimentin increased in TGF-β group;the expression levels of E-cadherin in the TGF-β+GGA group decreased,the expression levels of E-cadherin and vimentin in the GGA group decreased;Compared with TGF-β group,the expression level of E-cadherin increased,while the expression level of vimentin decreased in TGF-β+GGA group and GGA group;compared with the TGF-β+GGA group,the expression level of E-cadherin increased,while the expression level of vimentin decreased in GGA group,and the difference was all statistically significant(P<0.05).4.When A549 cells were treated with 50μmol/L GGA,the effect of HSP70 on cell morphology was observed: compared with the blank group,the cells in the TGF-β group were more elongated and fibrous;but when GGA was added alone,the cell edges became more rounded.When A549 cells were treated with 200μmol/L GGA,the effect of HSP70 induced by it on the cell morphology was observed: compared with the blank group,the cells of TGF-β group were more elongated and fibrous;compared with TGF-β group,the cell edges of TGF-β+GGA group cell were tended to be round and blunt,when GGA was added alone,the cells were more round and blunt.5.When A549 cells were treated with 50μmol/L GGA,the effect of HSP70 on migration ability in A549 cell was observed: compared with the blank group,scratch widths of TGF-β group and TGF-β+GGA group become more narrow,scratch widths of GGA group became wider;compared with TGF-β group,the scratch widths of both TGF-β+GGA group and GGA group became wider;compared with TGF-β+GGA group,the scratch width of GGA group became wider,and the difference all was statistically significant(P<0.05).When A549 cells were treated with 200μmol/L GGA,the effect of HSP70 induced by GGA on migration ability was observed: compared with the blank group,the scratch width of TGF-β group become more narrow,the scratch width of TGF-β+GGA group and GGA group became wider;compared with TGF-β group,both TGF-β+GGA group and GGA group had wider scratch widths,and the difference was both statistically significant(P<0.05).Conclusion 1.HSP70 inhibited TGF-β induced EMT progression in A549 cell.2.HSP70 changes TGF-β induced cell morphology and migration ability in A549 cells.3.HSP70 may be an important target in the EMT process. |
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