Effects And Mechanisms Of ATRX Deletion On Alveolar Epithelial Type Ⅱ Cell Damage By Irradiation

Radiation-induced pulmonary fibrosis（RIPF）is a serious complication of radiotherapy,mainly manifests as chronic lung function loss,and ultimately leads to the death of patients due to respiratory failure.RIPF causes chronic bleeding in the lungs,which is caused mainly by the loss of capillaries in the lungs,induces fibroblast aggregation,promotes collagen releasing,and increases the deposition of extracellular matrix.A variety of cells and cytokines jointly participate in the regulation of the occurrence of RIPF,and the occurrence of RIPF are mainly involved in the alveolar epithelial cells,which were divided into alveolar epithelial typeⅠcells and alveolar epithelial typeⅡcells.The alveolar epithelial typeⅡcells account for only 7%of the alveolar surface area,but 15%of the total parenchymal cells.In recent years,the main viewpoints about the involvement of alveolar epithelial typeⅡcells in the occurrence of RIPF focus on:increasing apoptosis,decreasing proliferation,reducing the tendency of the transformation of typeⅡcells into typeⅠcells and inducing subsequent fibrosis,and the cell senescence releases a large number of pro-inflammatory factors to increase pulmonary fibrosis,and so on.Epithelial-mesenchymal Transition（EMT）is an important cell source of fibroblasts,and EMT can promote the transformation of alveolar epithelial typeⅡcells into fibroblasts.The alpha-thalassemia/mental retardation syndrome X-linked（ATRX）gene plays an important role in gene expression,DNA damage repair and replication,and the protein acts as a chromosomal remodeling protein.ATRX mutations are present in a variety of tumors and have also been found in lung cancer cells.Therefore,it has great significance to explore the role and mechanism of ATRX deletion on alveolar epithelial typeⅡcell damage by radiation and to reduce the occurrence of RIPF.Relevant literature and previous studies have shown that ATRX deletion could decrease DNA repair,induce cycle checkpoint activation,and regulate cell senescence and apoptosis.Moreover,ATRX mutations affect cell cycle regulation and DNA damage repair.The role and mechanism of ATRX function loss on damage of typeⅡalveolar epithelial cells by radiation and the occurrence of RIPF during lung cancer radiotherapy remain unclearly.In this study,the alveolar epithelial typeⅡcell model of silencing ATRX was used to investigate the damage effect and mechanism of ionizing radiation,aiming to prevent the occurrence of RIPF and to reduce degree of pulmonary fibrosis by radiation,thereby,to enhance the application in radiotherapy of tumors and to reduce the complications caused by clinical radiotherapy.Objective:To explore the effects of radiation on morphology,migration,proliferation and EMT in alveolar epithelial typeⅡcells,furthermore,to investigate the effects and molecular mechanisms of radiation mediated by ATRX deletion on EMT,proliferation,migration,cell cycle distribution,DNA damage repair,apoptosis and senescence.Therefore,it provides a new explanation for the occurrence of RIPF and a new biological theory for clinical radiotherapy.Methods:1.The non-target control and three GIPZ?sh RNA plasmids targeting ATRX were transfected into 293T cells,respectively,they were sh Control,sh ATRX1,sh ATRX2 and sh ATRX3.After lentivirus were packaged,A549 cells were infected to construct stable cell model silenced ATRX,and to identify whether the model were constructed successfully by Western blot and IF,and cell models were named NC and ATRX^low.2.Morphological changes of Beas-2b and A549 cells were observed by optical microscopy at 0,12,24 and 48 h after 0,2 and 10 Gy irradiation.3.The expression changes of cytoskeleton protein（F-actin）in Beas-2b and A549cells,NC and ATRX^low cells were observed by immunofluorescence method after irradiation with 0 and 10 Gy.4.The changes of cell migration ability of Beas-2b and A549 cells,NC and ATRX^low cells were measured by would healing assay after irradiation with 0,2 and 10Gy.5.The changes of cell proliferation in Beas-2b and A549 cells,NC and ATRX^lowcells were detected by CCK8 reagent after 0,2 and 10 Gy irradiation.6.EMT-related proteins in Beas-2b and A549 cells,NC and ATRX^low cells were detected by Western Blot after irradiation with 0,2 and 10 Gy.7.Immunofluorescence was used to detect the number ofγH2AX foci in NC and ATRX^low cells at 0.5,1,3,6 and 12 h after 0 and 10 Gy irradiation,and the expressions of DNA damage proteins were detected by Western Blot.8.The apoptosis rate of NC and ATRX^low cells was determined by flow cytometry using Annexin V-PE/7-ADD apoptosis Kit after irradiation with 0,2 and 10 Gy.9.The cycle percentage changes of NC and ATRX^low cells were detected by flow cytometry with PI staining after irradiation with 0,2 and 10 Gy,and the expression of related cycle proteins were detected by Western Blot.10.Theβ-galactosidase cell senescence detection kit was used to detect the changes of cell positive rate of NC and ATRX^low cells at 0,0.5,1,2 and 6 d after 0 and10 Gy irradiation.11.Gene differential expression profiles of NC and ATRX^low cells were detected by RNA-seq technique after irradiation with 0 and 10 Gy.12.Statistical analysis:SPSS 24.0 software was used for statistical analysis,and the experimental data were expressed as mean±standard deviation（x±s）.The t test of two independent samples was used,and P<0.05 was considered to be statistically significant.Statistical graphs were drawn using Graph Pad Prism 8 software.Results:1.Effects of radiation on morphology,EMT markers,migration and proliferation of Beas-2b and A549 cells（1）The effect of radiation on the morphology in Beas-2b and A549 cellsThe morphology in Beas-2b and A549 cells changed after 0,2 and 10 Gy irradiation at 0,12,24 and 48 h.With irradiation dose increasing,the A549 cells changed from the original epithelioid cell morphology which was oval and tightly arranged without direction to the interstitial cell morphology which was long and loosely distributed with fusiform direction.The same pattern was also found in Beas-2b cells,but after irradiation,Beas-2b cells showed obvious trailing,shrinking and dying.The changes of F-actin in the cytoskeleton of Beas-2b and A549 cells were observed by immunofluorescence after 0 and 10 Gy irradiation,and the results showed that the F-actin expression in Beas-2b and A549 cells increased.It is suggested that radiation can cause morphologic changes of alveolar epithelial typeⅡcells.（2）Effect of radiation on EMT markers in Beas-2b and A549 cellsWestern Blot was used to detect the changes of EMT markers in Beas-2b and A549cells at 24 and 48 h after 0,2 and 10 Gy irradiation.The results showed that the expression levels of N-cadherin,Vimentin andα-SMA in Beas-2b and A549 cells were increased after irradiation,indicating that radiation can induce EMT in lung alveolar epithelial typeⅡcells.（3）Effect of radiation on migration of Beas-2b and A549 cellsThe changes of migration ability of Beas-2b and A549 cells at 0,24 and 48 h after0,2 and 10 Gy irradiation were detected by scratch assay.The results showed that the migration ability of A549 cells was significantly enhanced after 2 Gy irradiation compared with that of 0 Gy group（P<0.01）,the same trend was observed in Beas-2b cells,but the difference was insignificant.After 10 Gy irradiation,the migration ability of A549 cells was significantly enhanced（P<0.01）,but the migration ability of Beas-2b cells was not significantly changed after irradiation.The results showed that ionizing radiation could induce EMT in lung epithelial cells and enhance cell migration.It is suggested that radiation can induce EMT in alveolar epithelial typeⅡcells,and EMT can promote cell migration.（4）The effect of radiation on the proliferation of Beas-2b and A549 cellsCCK8 results showed that the proliferation in Beas-2b and A549 cells was significantly inhibited after 2 and 10 Gy irradiation compared with 0 Gy irradiation（P<0.05）,and at 24 h after irradiation,with the dose increasing,the proliferation inhibition effect was more significant（P<0.05）.It is suggested that radiation damage on alveolar epithelial typeⅡcells can reduce the cell proliferation.2.Construction of A549 cell model targeting silencing ATRXAfter lentivirus were packaged using 293T cells,A549 cells were infected,the expression of GFP protein were observed under fluorescence microscopy,suggesting sh RNA fragments were carried into cells,and Western Blot results showed that ATRX expressed in A549 cells and cells of sh Control,but it expressed less in cells of sh ATRX1,sh ATRX2,sh ATRX3,suggesting that alveolar epithelial typeⅡcell models stably silenced ATRX were constructed successfully,named NC and ATRX^low.3.Effects of silencing ATRX and radiation on A549 gene expression profileRNA-seq was used to detect the effects of silenced ATRX and 10 Gy irradiation on gene expression in A549 cells.The results showed that compared with the control group,429 genes were up-regulated and 254 genes were down-regulated in ATRX^lowcells at 0 Gy,and a total of 683 genes were differentially expressed.After 10 Gy irradiation,the expressions of 530 genes were up-regulated and 328 genes were down-regulated,and 858 genes were differentially expressed.KEGG Pathway enrichment analysis of differential genes showed that silencing ATRX induced gene expression differences in A549 cells mainly involved in cell cycle regulation,DNA replication,lipid metabolism,redox reaction,positive regulation of cell proliferation and epithelial differentiation.Gene expression differences in A549 cells induced by silencing ATRX and 10 Gy irradiation were mainly manifested in biological processes such as translation,oxidative stress,ATP biosynthesis,proton transmembrane transport and apoptosis.By analyzing the KEGG Pathway annotation classification results of the differential genes,the results showed that the differentially expressed genes were involved in the pathways associated with ECM,inflammatory response and fibrosis progression.4.Effect of radiation on EMT markers and morphology of A549 cells silenced ATRX（1）Effect of radiation on EMT markers on A549 cells silenced ATRXAfter irradiation by 0,2 and 10 Gy in NC and ATRX^low cells,at 24 and 48 h,the protein expressions of EMT-related markers N-cadherin,Vimentin andα-SMA were measured.Western Blot results showed that the expressions of N-cadherin,Vimentin andα-SMA in ATRX^low cells increased after 2 Gy irradiation compared with NC cells.At 24 h,the expression of N-cadherin andα-SMA increased significantly,while the expression of Vimentin did not change significantly.At 48 h,the expression of N-cadherin,Vimentin andα-SMA increased significantly.After 10 Gy irradiation,the expressions of N-cadherin,Vimentin andα-SMA in ATRX^low cells increased significantly,indicating a same pattern at 24 h and 48 h.It is suggested that silencing ATRX can enhance the occurrence of radiation-induced EMT in A549 cells.（2）Effect of radiation on cytoskeleton in A549 cells silenced ATRXThe changes of cytoskeleton protein F-actin in NC and ATRX^low after 0 and 10 Gy irradiation were observed by immunofluorescence method（IF）,and the results showed that the expressions of F-actin in NC and ATRX^low increased and it wa more obvious in ATRX^low cells.These results suggested that silencing ATRX can promote the expression of F-actin in A549 cells and the development of interstitial cell morphologic changes,furthermore to enhance the radiation-induced EMT.5.Effect of radiation on the migration of A549 cells silenced ATRXCompared with NC cells,the migration ability of ATRX^low cells increased significantly at 24,48 and 72 h after 0 Gy irradiation（P<0.05）;the migration ability of ATRX^low cells increased significantly at 24 and 72 h after 2 Gy irradiation（P<0.05）,but there was no significant change at 48 h;at 24,48 and 72 h after 10 Gy irradiation,the migration ability of ATRX^low cells increased significantly（P<0.01）.Compared with 0 Gy,ATRX^low cell migration increased significantly at 48 h after 2 and 10 Gy irradiation（P<0.01）.It is suggested that the migration ability of A549 cells is enhanced after ATRX silencing,and radiation can enhance these effects.6.Effect of radiation on the proliferation in A549 cells silenced ATRXCell proliferation was detected by CCK8 reagent,which was expressed by the absorbance value A₄₅₀.The results showed that compared with NC cells,the A₄₅₀ value in ATRX^low cells decreased significantly at 24 h after 0 Gy（P<0.05）;at 24 h after 2and 10 Gy irradiation,the A₄₅₀ value in ATRX^low cells decreased significantly（P<0.05）.Compared with 0 Gy,A₄₅₀ value in ATRX^low cell s decreasedignificantly at 48 h after 2and 10 Gy irradiation（P<0.01）.It was suggested that the proliferation of A549 cells decreased after ATRX silencing,and the cell proliferation could be inhibited at the early stage after irradiation,but it could be promoted with the time prolongation.7.Effects of radiation on DNA damage repair in A549 cells silenced ATRXThe changes ofγH2AX foci in NC and ATRX^low cells after 10 Gy irradiation were observed by IF.The results showed thatγH2AX foci in both groups began to increase after irradiation,and reached the maximum at 1 h,and then began to decrease.Compared with NC cells,theγH2AX foci in ATRX^low cells was not significantly different at 0.5 and 1 h after irradiation,but increased significantly at 3,6 and 12 h（P<0.01）.8.Effect of irradiation on cycle progression A549 cells silenced ATRXFlow cytometry was used to detect the cell cycle progression.The results showed that compared with NC cells,the percentage of G₀/G₁ decreased and the percentage of S increased significantly at 48 h after 0 Gy irradiation（P<0.01）;at 24 h after 2 Gy irradiation,the percentage of G₀/G₁ increased in ATRX^low cells,suggesting G₀/G₁ arrest,and the percentage of S decreased significantly（P<0.01）.Compared with 0 Gy,at 24h after 10 Gy irradiation,the percentage of G₀/G₁ and S was lower in NC cells and ATRX^low cells,while the percentage of G₂/M phase increased,suggesting G₂/M arrest,but there was no significant difference between both of them.The expression of cell cycle-related proteins was detected by Western Blot.The results showed that the expression of p53 and p21 proteins increased in NC cells and ATRX^low cells after 2 and 10 Gy irradiation compared with 0 Gy irradiation.Compared with NC cells,the protein expression of p53 and p21 increased in ATRX^low cells at 24h.Cyclin D1 and CDK4 proteins involved with G₁/S phase reduced significantly in ATRX^low cells compared with NC cells at 24 and 48 h after 2 Gy irradiation.9.Effects of radiation on apoptosis of A549 cells silenced ATRXFlow cytometry was used to detect the apoptosis rate in NC cells and ATRX^lowcells.The results showed that the apoptotic rate in NC and ATRX^low cells increased after2 and 10 Gy irradiation.Compared with NC cells,the apoptotic in ATRX^low cells did not change significantly after 0 and 2 Gy irradiation.Apoptotic rate in ATRX^low cells increased significantly at 24 h after 10 Gy irradiation（P<0.05）.It is suggested that radiation can promote the apoptosis of A549 cells silenced ATRX,but there is a difference depending on the dose.10.Effects of radiation on senescence in A549 cells silenced ATRXβ-galactosidase staining staining was used to detect cell senescence.The results showed that the positive rate in NC cells and ATRX^low cells increased gradually,but there was no significant difference both of them.At 1,2 and 6 d after 10 Gy irradiation,the senescence positive rate in ATRX^low cells was significantly higher than that in NC cells（P<0.01）;moreover,compared with 0 Gy,the senescence positive rate in ATRX^low cells at 2 and 6 d after 10 Gy irradiation increased significantly（P<0.01）,suggesting that radiation could promote cell senescence in A549 cells silenced ATRX.Conclusions:1.Radiation can induce the epithelial-mesenchymal transformation（EMT）of alveolar epithelial typeⅡcells,mainly manifesting as morphological enlargement,increasing expression of skeleton protein F-actin,and enhancing cell migration ability,which may promote the occurrence of RIPF.2.In this study,we successfully constructed the alveolar epithelial typeⅡcells A549 model targeting silencing ATRX.Ionizing radiation reduces the cell DNA damage repair ability and activates the ATM-Chk2-p53 pathway to mediate G₀/G₁ phase arrest.3.Silencing ATRX can enhance the radiation-induced senescence and apoptosis of alveolar epithelial typeⅡcells,and thus reduce cell proliferation.However,the difference in dose may be one of the reasons for radiation-induced pulmonary fibrosis.4.Radiation can enhanced EMT inⅡtype alveolar epithelial cells after ATRX silencing,which is involved in the activation of the p53-mediated TGF-β/Smads pathway;5.Silencing ATRX can induce changes in the expression of signaling pathways related to immune system,DNA damage and fibrosis at the transcriptional level in alveolar epithelial typeⅡcells,and thus promote the occurrence of EMT.