The Stemness Transformation Of CD133~-lung Adenocarcinoma Cells Induced By Ionizing Radiation And Its Underlying Mechanisms

Objective Cancer stem cells(CSCs)possess intrinisic resistance to radiation,and are responsible for cancer recurrence and metastasis after radiotherapy.Many studies have proved that non-stem(adult)cancer cells could dedifferentiate into CSCs after undergoing epithelial-mesenchymal transition(EMT).Our previous work showed that ionizing radiation could induce lung adenocarcinoma cells(A549)to undergo EMT,These results suggest that ionizing radiation may transform non-stem A549 cells into stem A549 cells.We will study on the effect of ionizing radiation on stem characteristics of non-stem lung adenocarcinoma cells and the role of IL-6/JAK2/STAT3 signaling pathway in radiation-induced stemness transformation of non-stem lung adenocarcinoma cells.Methods The CD133-cells were sorted from A549 cells by using magnetic activated cell sorting(MACS).CD133-cells were irradiated by X-ray and CD133 positive cells in CD133-cells were detected by flow cytometry,the sphere formation in CD133-cells were detected by sphere formation assay,and the migration abilities in CD133-cells were detected by transwell migration assay.The SOX2 and Oct4 protein in CD133-cells were detected using Western blot after CD133-cells were treated X-ray.Further,the Western blot was used to detect the expression of IL-6 and phosphorylated STAT3(p-STAT3)protein in CD133-cellsafter 2 Gy and 4 Gy X-ray.CD133-cells were stimulated with 10,20,40 ng/ml IL-6 in vitro to detect the stemness properties of CD133-cell;IL-6/JAK2/STAT3 signal induced by X-ray irradiation was blocked by JAK2 kinase inhibitor AG490 pathway activation detects changes in CD133-cell stemness properties.Statistical analysis was performed using GraphPad Prism 5 softerware,and P was interpreted as possess differences of statistical significance.Results 1、The effect of ionizing radiation on the stemness properties of CD133-lung adenocarcinoma cellsAfter 2 Gy and 4 Gy X-ray irradiation,the proportion of CD133-cells was increased significantly;the sphere formation and migration ability of CD133-cells increased in a dose-dependent manner and the difference was statistically significant(P<0.05);After 24 hour and 48 hours of 2 Gy X-ray irradiation,the SOX2 and Oct4 protein in CD133-cells was significantly up-regulated and time-dependent;Above experimental results suggest that a certain dose of ionizing radiation can induce stemness properties of CD133-lung adenocarcinoma cells.2、The role of IL-6/JAK2/STAT3 in the stemness transformation of CD133-lung adenocarcinoma cells induced by ionizing radiationAfter irradiation with X-ray at dose of 2 Gy and 4 Gy,IL-6 and p-STAT3 protein in CD133-cells were significantly increased in a dose-dependent manner.Similar to X-ray irradiation,the stimulation of IL-6 significantly up-regulated p-JAK2 and p-STAT3 levels,and the sphere formation,migration ability and SOX2、Oct4 protein of CD133-cells in a dose-dependent manner.the adding of JAK2 inhibitor(AG490)in the culture significantly suppressed p-STAT3 actication that induced by IL-6 and the expression of SOX2、Oct4 and the ability of sphere formation was also inhibited.Similarly,AG490 also significantlyinhibited the expression of p-STAT3、Oct4 and the ability of sphere formation that induced by 2 Gy X-ray.Conclusion Ionizing radiation induce stemness in CD133-(adult)lung adenocarcinaoma cells to acquire cancer stem cell properties,and its mechanism is related to ionizing radiation activated though IL-6/JAK2/STAT3 signaling pathway...