The Role Of STAT3 In Premature Senescence Of Lung Adenocarcinoma Induced By X-ray Irradiation

ObjectiveThe discovery of premature aging induced by X-rays,making research on cell-related molecules are becoming a hotspot and a new research direction for tumor radiotherapy.It is worth noting that the exact mechanism of cell radioresistance has not been elucidated in non-small cell lung cancer.In our previous studies,we found that premature aging of lung adenocarcinoma A549 cells can be induced and STAT3 can be activated by X-ray irradiation.on the basis of this,to explore further the role of STAT3 in the induction of premature senescence of lung adenocarcinoma A549 cells after X-ray irradiation,to study the STAT3 promoting X-ray-induced premature aging of lung adenocarcinoma A549 cells signal pathway,to explore potential strategies for the treatment of lung adenocarcinoma from the perspective of premature cell,it have important value and significance on basic research and expected clinical research in tumor radiotherapy.Method1.2 Gy,4 Gy,8 Gy X-rays irradiated A549 cells,after 24 h,48 h and 72 h respectively,the cell proliferation rate was measured by MTT assay,senescent cells were detected by ?-galactosidase staining assay,and the Lyso-Tracker fluorescent probe was used to detect and analyze the change of lysosome quantity;2.Used Western Blot to detect the expression of STAT3,p-STAT3,BECN1,CAV 1,p62,p53 and pl6 proteins in A549 cells after 4 Gy X-ray irradiation.3.After transfecting A549 cells with the overexpression plasmid pcDNA3.0-STAT3 for 36 h,pre-aging of A549 cells was detected by ?-galactosidase staining assay,the positive rate of premature senescence cells was counted.4.After treatment of A549 cells with AG490 and RAPA inhibitor for 24 h,2 Gy,4 Gy,and 8 Gy X-rays were irradiated to A549 cells.respectively.The premature aging of A549 cells was detected by ?-galactosidase staining test at 72 h and the positive rate of premature senescence cells was counted.The expression levels of STAT3,p-STAT3,BECN1,CAV 1,p62.p53 and p16 proteins after overexpression of STAT3 were detected by Western Blot.Results1.2 Gy,4 Gy and 8 Gy X-rays were irradiated to A549 cells,respectively.At 24'h,48 h and 72 h,the cell proliferation rate decreased significantly(P<0.05).The(3-galactosidase staining experiment showed that there was a significant time dose effect on the proportion of premature aging cells(P<0.05).After irradiation,the cell morphology exhibits a flat and enlarged phenotype,including irregular shapes,and the cytoplasm contains a large number of vacuoles.Lyso-Tracker fluorescent probes were used to detect A549 cells irradiated by X-rays.It was found that the number of lysosomes increased significantly after exposure to 2 Gy,4 Gy and 8 Gy X-rays at 48 h(P<0.05).2.4 Gy X-rays irradiated A549 cells.At 24 h,48 h and 72 h,it was found that the phosphorylation level of STAT3 protein was significantly increased.;After overexpressing STAT3,the ?-galactosidase staining experiment showed that the proportion of premature aging cells increased significantly(P<0.05),and overexpression of STAT3 caused an increase in the number of A549 premature aging cells.and the phosphorylation level of STAT3 protein was significantly increased;After A549 cells are treated with AG490 and RAPA inhibitors,the ?-galactosidase staining experiment showed that the proportion of premature aging cells decreased significantly(P<0.05),inhibition of STAT3 caused a decrease in the proportion of A549 premature aging cells.Compared with the simple irradiation group,the phosphorylation level of STATS protein was significantly decreased.X-ray induced premature senescence of A549 cells is dependent on the activation of STATS.3.4 Gy X-rays irradiated A549 cells.At 24 h,48 h and 72 h.it was found that the phosphorylation level of STAT3 protein was significantly increased,the expression of BECN1 protein was up-regulated;After overexpressing STAT3,the expression of BECN1 protein was increased;After A549 cells are treated with AG490 and RAPA inhibitors,the expression of BECN1 protein was down-regulated,and the expression of p62 protein was decreased.The pathway of X-ray induced premature senescence of A549 cells is the positive regulation of BECN1 expression by STAT3,inhibition of p62 protein expression,and induction of premature senescence.4.4 Gy X-rays irradiated A549 cells.At.24 h,48 h and 72 h,it was found that the phosphorylation level of STAT3 protein was significantly increased,the expression of CAV 1 protein was down-regulated,p53 protein was up-regulated;After overexpressing STAT3,the expression of CAV 1 protein was decreased,the expression of p53 protein were up-regulated.The pathway by which X-rays induce premature aging of A549 cells may be STAT3 negatively regulates CAV1 and promotes p53 protein expression.This pathway needs to be further determined.5.4 Gy X-rays irradiated A549 cells.At 24 h,48 h and 72 h,it was found that the phosphorylation level of STAT3 protein was significantly increased,pl6 protein was up-regulated;After overexpressing STAT3,the expression of p16 protein were up-regulated.After A549 cells are treated with AG490 and RAPA inhibitors,the expression of p62 protein was decreased.The pathway of X-ray induced premature senescence of A549 cells is that STAT3 positively regulates p16 expression and promotes premature senescence of A549 cells.Conclusion1.2 Gy,4 Gy,8 Gy dose X-ray irradiation induced premature aging of A549 cells,and X-ray induced premature senescence of A549 cells is dependent on the activation of STATS.2.STAT3-mediated X-ray induced premature senescence of A549 cells may involve three pathways.X-ray may induce premature aging of A549 cells by STAT3-BECN1-p62;X-ray may induce premature aging of A549 cells by STAT3-CAV 1-p53.which needs further verification;X-ray may induce premature aging of A549 cells by STAT3-p16.