Study On Dedifferentiation Of Lung Adenocarcinoma Non-Cancer Stem Cells Into Cancer Stem Cells Induced By Ionizing Radiation And Its Potential Mechanism

Objectives:Lung adenocarcinoma(LUAD)is the main pathological subtype of lung cancer.Radiotherapy is one of the main treatments for patients with LUAD,after radiotherapy,the tumor will shrink or even disappear,but most of them will still have recurrence or metastasis.Some previous studies have shown that cancer stem cells(CSCs)which have natural resistance to radiation are the key factors leading to tumor recurrence and metastasis after radiotherapy.In addition,some studies have found ionizing radiation could even induce some non-CSCs with potential stemness dedifferentiation into CSCs,but this phenomenon has not been studied in LUAD.This study is the first to explore whether ionizing radiation can induce non-CSC dedifferentiation into CSC in LUAD,and to further study the possible mechanism,which is of great significance to better explain the mechanism of radiation resistance and to find potential targets that can predict and improve the radiosensitivity of patients with LUAD.Methods:This study was divided into two parts:1.Can ionizing radiation induce non-CSC dedifferentiation into CSC in LUAD?Firstly,the radioresistant A549 cell(AR)was constructed,and the expression of stemness-related proteins(Oct4,Nanog,Sox2,and CD133)and the CSC sphere formation ability in vitro were detected.Then,the CD 133 antibody-coupled magnetic beads were incubated with A549 and H1299 cells,and the CD133+cells were removed by magnetic cell sorting to obtain a CD133-/low cell population.The CD133-/low cells were irradiated with 0,2,4 and 8Gy X-ray,respectively.The changes of stemness-related genes(oct4,nanog,sox2)and their protein expression were detected at different time points after irradiation.Also,the proportion of CD133+ cells was detected by flow cytometry and the CSC sphere-formation ability was observed by sphere-formation assay in the surviving cells after irradiation.2.Study on the mechanism of regulating the dedifferentiation of non-CSC after ionizing radiation in LUADFirstly,to study the role of epithelial-mesenchymal transition(EMT)in non-CSC plasticity after ionizing radiation and radioresistance of LUAD,the expression differences of EMT-related proteins(E-cadherin,N-cadherin and Vimentin)in the surviving cells of CD133-/low A549(A-)and CD133-.low H1299(H-)after X-ray irradiation with different doses were detected by Western blot.Then the group with the most obvious dedifferentiation changes in the first part of the study were sequenced in mRNA level,and the unirradiated cells were used as controls to find the key genes and pathways regulating the dedifferentiation of non-CSC after radiation.The experiments in first part of this study were repeated after the expression of the selected gene was restored to observe whether the gene reverses the non-CSC dedifferentiation of LUAD induced by ionizing radiation.Results:1.Study on non-CSC dedifferentiation of LUAD(1)Compared with parent A549 cell(AP),the expression of stemness-related proteins in AR group were significantly up-regulated,and the sphere formation ability of AR group was also significantly increased.(2)The stemness-related genes of A-and H-cells were up-regulated after X-ray irradiation,and the changes became more obvious with the increase of radiation dose,especially at 96 h after 8Gy irradiation.The expression of stemness-related proteins showed a similar trend and was significantly up-regulated at 96 h after 4Gy or 8 Gy X-ray irradiation.(3)The proportion of CD133+ cells increased significantly in the surviving cells of Aand H-cells at 96 h after 8 Gy X-ray irradiation.(4)The sphere formation ability in the surviving cells of A-and H1 cells at 96h after different doses of X-ray irradiation increased in a dose-dependent manner,especially in 8Gy group.The frequency of forming CSC spheres in the surviving cells at 96 h after 8 Gy X-ray irradiation was significantly increased,which was about 5 times higher than that in the unirradiated group.2.Study on the mechanism of regulating the dedifferentiation of non-CSC after ionizing radiation in LUAD(1)The expression of E-cadherin protein in A-and H-cells was down-regulated after different doses of X-ray irradiation,and the expression of N-cadherin protein was also down-regulated,most obviously after 8Gy irradiation,while the expression of Vimentin protein had an up-regulated trend,and the change was most obvious after 4Gy irradiation.Compared with AP group,the expression of E-cadherin protein was down-regulated,and the expression of N-cadherin and Vimentin protein was up-regulated in AR group.(2)Gene transcriptome sequencing and bioinformatics were predicted that EGR1 may be the key gene that regulates non-CSC plasticity of LUAD induced by ionizing radiation.EGR1 gene and protein were up-regulated in residual A-and H-cells at 96h after 8Gy X-ray irradiation.At the same time,EGR1 protein expression was up-regulated in A549 and H1299 cell-derived CSC spheres and in AR cells.(3)Knockdown of EGR1 expression can inhibit the up-regulation of stemness-related protein expression,the increase in the proportion of CD133+cells and the increase in sphere formation ability in A-and H-cells induced by X-ray irradiation.Moreover,knockdown of EGR1 expression can restore the radiosensitivity of radioresistant A549 cell.(4)Through database prediction analysis,GDF15 may be a key target gene for EGR1 to regulate non-CSC dedifferentiation of LUAD cells post-radiation.At the same time,GDF15 protein was upregulated in A549 and H1299 cells derived from CSC spheres and radioresistant A549 cells.(5)Knockdown of EGR1 expression can cause down-regulation of GDF15 expression,which indicates that EGR1 positively regulates GDF15 expression.At the same time,GDF15 expression was significantly up-regulated in the residual A-and Hcells at 96h after 8Gy X-ray irradiation,and the secretion of GDF15 protein in the supernatant of the culture medium was significantly increased.Conditioned medium derived from A-and H-cells cultured for 96h after 8Gy X irradiation reversed the inhibitory effect of EGR1 knockdown on non-CSC dedifferentiation of LUAD cells induced by ionizing radiation to a certain extent,and the effect became stronger with the increase of the amount of conditioned medium,showing a certain dose dependence.Conclusions:1.The increase of stemness after ionizing radiation may lead to radioresistance of A549 cell.2.X-ray irradiation can induce the dedifferentiation of LUAD cells from non-CSC to CSC to a certain extent in a dose-and time-dependent manner,with the most obvious change at 96h after 8Gy irradiation.3.EMT may not dominate non-CSC dedifferentiation in LUAD cells induced by irradiation,but it may mediate radiation resistance of A549 cell.4.The up-regulation of EGR1 may induce radioresistance of A549 cell.5.Ionizing radiation may induce non-CSC dedifferentiation of LUAD by up-regulating EGR1/GDF15 signal axis.6.GDF15 may be used as a secretory protein to regulate the dedifferentiation of non-CSC in post-radiation LUAD cells by autocrine or paracrine,which needs further research to verify.