The Study Of Chemokine CCL19 Promotes Invasion And Metastasis Of A549 Through Regulating AKT Signal Pathway

[Objective]:To explore the role and mechanism of CCL19 in epithelial mesenchymal transition and invasion,metastasis of lung adenocarcinoma cells.[Method]:(1)The experimental groups were as follows:blank control group(A549 cell),A549 cell plus DMSO group,A549 cell plus CCL19 group,A549 cell plus MK2206 group,A549 cell plus CCL19 and MK2206 group.(2)CCK8 assay was used to detect the proliferation ability of lung cancer cells in different groups.(3)After establishing an anoikis model,flow cytometry was used to detect the anoikis of lung cancer cells in different groups.(4)Scratch test was used to detect the migration ability of different groups of lung cancer cells.(5)Transwell assay was used to detect the invasion ability of lung cancer cells in different groups.(6)WB assay was used to detect the expression of pan-AKT,p-AKT(Thr308),p-AKT(Ser473),snail,N-cad and E-cad.(7)Immunohistochemical staining was used to detect the expression of MMP-9 and CCL19 in different area of clinical lung cancer tissues.(8)GraphPad Prism 6 statistical software carries on the statistical analysis to the data.[Result]:(1)CCK8 test found that CCL19 did not affect the proliferation of A549 cell(P>0.05),and the proliferation of cell was significantly inhibited after added MK2206(P<0.05).(2)Flow cytometry found that after the addition of CCL19,the anoikis was inhibited obviously(P<0.05).After only adding MK2206,the anoikis was not inhibited(P>0.05),while after both CCL19 and MK2206 were added,the anoikis was also obviously inhibited(P<0.05).(3)The scratch test showed that CCL19 significantly enhanced the migration ability of A549 cell,while the migration ability of A549 cell reduced significantly after adding MK2206(P<0.05).(4)Transwell test found that after adding CCL19,the invasion ability of A549 increased significantly(P<0.05),while the invasion ability decreased significantly after adding MK2206(P<0.05).(5)WB test found that CCL19 could enhance the expression of p-AKT(Thr308),p-AKT(Ser473),snail and N-cad and reduce the expression of E-cad.After adding MK2206,the expression of p-AKT(Ser473),snail and N-cad decreased,and the expression of E-cad increased.(6)The expression of MMP-9 and CCL19 in lung cancer tissues of different regions were closely related to lymph node metastasis,not related to the pathological type,and there was no regional difference.[Conclusion]:(1)CCL19 can inhibit the occurrence of anoikis in A549 cell and promote its migration and invasion,but has no obvious regulating effect on cell proliferation.(2)MK2206 can inhibit the migration and invasion of lung cancer cells to a certain extent,after adding CCL19 the expression level of p-AKT is enhanced,this shows that the PI3K/AKT signal transduction pathway plays an important role in the process of CCL19 promoting the migration and invasion of lung cancer cells.This may be related to after CCL19 combined with its receptor CCR7 that it activates the PI3K/AKT and other signal transduction pathways,and eventually activates the expression of related genes;CCL19 can promote epithelial mesenchymal transition of A549 cell,while MK2206 can inhibit the role of CCL19 to promote the epithelial mesenchymal transition in A549 cell in a certain extent;(3)The expression of MMP-9 and CCL19 is related to lymph node metastasis,is not related to pathological type of lung cancers,and there is no regional difference.