Effects And Mechanisms Of Silencing ATRX On Cell Cycle Arrest Induced By Ionizing Radiation

Nowadays,cancer is a major problem endangering public health.Although the overall mortality rate is declining,it is still the biggest killer of human death.Radiation therapy is an important treatment for tumors,which can cause cell death by destroying the cell cycle process,so as to achieve the purpose of treating tumors.The direct target of radiation therapy is DNA,and cell cycle arrest is mainly to gain time for DNA damage repair,the mechanism of DNA damage repair is considered to be the Achilles'heel.Radiation can cause cell cycle arrest,which is mainly to gain enough time for DNA damage repair,and when serious DNA damage induced by radiation can not repair,thus apoptosis and senescence will be developed..The?-thalassemia/mental retardation syndrome X-linked gene is a key factor in chromatin remodeling and participates in DNA methylation,DNA repair,and telomere stabilization and recruitment of histone variant H3.3 and so on.In this study,a stable tumor cell model silenced ATRXwas established,after ionizing radiation is administered,the effects on cell cycle arrest,DNA damage repair,apoptosis and senescence,and radiation sensitization and molecular mechanisms were explored,this experiment also provided the theoretical and experimental data for radiosensitization targets.Objective:To investigate the effects and molecular mechanisms of silencingATRX on ionizing radiation-induced tumor cell cycle arrest,furthermore,to explore the effects on DNA damage repair,cell senescence and apoptosis,radiosensitivity and relative molecular mechanisms,to provide theoretical and experimental data for molecular targets of radiosensitization..Methods:1.Three shRNA sequences targeting ATRX were designed and cloned into the lentiviral vector pGIZ,293T cells were used to package lentivirus and infect cervical cancer HeLa cells,and silencing efficiency was measured using immunofluorescence(IF)and Western blotting,and the non-target sequences were used for controls.;On this basis,the lentivirus of shATRX continued to infect colon cancer HCT116p53wild-type and knock-out cells,shCon-p53+/+,shATRX-p53+/+,shCon-p53-/-and shATRX-p53-/-were named,Western blotting was used to verify the silencing efficiency of the cell model.2.X-ray irradiation conditions:X-RAD 320i X deep irradiator was used for irradiation under the following conditions:voltage 180 kV,current 20 mA,target distance 70 cm,dose rate 1.0 Gy/min;3.ShCon-p53+/+,shATRX-p53+/+,shCon-p53-/-and shATRX-p53-/-cells were detected by flow cytometry at 24 and 48 h after 2 and 8 Gy irradiation;WB was used to detect the expression changes of related cyclins;4.After shCon-p53+/+,shATRX-p53+/+,shCon-p53-/-and shATRX-p53-/-cells were irradiated with 2 and 8 Gy,the changes of cell proliferation were detected by CCK8 reagent;5.After shCon-p53+/+,shATRX-p53+/+,shCon-p53-/-and shATRX-p53-/-cells were irradiated with 0,2,4,6 and 8 Gy,the survival fraction was calculated by clone formation,and dose survival curve was drawn to judge the change of cell sensitivity;6.shCon-p53+/+,shATRX-p53+/+,shCon-p53-/-and shATRX-p53-/-cells were collected at 0,3,6 and 24 h after 8 Gy irradiation,and immunofluorescence technology was used to detect?H2AXfluorescence intensity with Rad51 fluorescence intensity,and Wblotting was used to detect the expression of DNA damage repair proteins;7.ShCon-p53+/+,shATRX-p53+/+,shCon-p53-/-and shATRX-p53-/-cells were collected at 0.5,1,2 and 4 days after 4 Gy irradiation,SA-?-gal kits were used to detect changes in cell senescence;8.ShCon-p53+/+,shATRX-p53+/+,shCon-p53-/-and shATRX-p53-/-cells were stained with Annexin V and 7-AAD apoptosis reagents at 24 and 48 h after 2 and 8Gy irradiation.Afterwards,flow cytometry was used to detect changes in apoptotic rate;9.The shCon-p53+/+and shATRX-p53+/+cells were recruited 24 hours after 8Gy,using immunoprecipitation(IP)technology to explore the interaction between ATRX,Daxx and p53.10.Statistical processing:SPSS 24.0 software was used for statistical analysis,and the data were expressed in the form of mean±standard deviation(x±s),and the comparison of sample mean between multiple groups was by one-way ANOVA.When the p value was less than 0.05 or 0.01,the difference was considered statistically significant.Results:1.Successfully constructed HCT116(p53+/+and p53-/-)cell models targeting-silenced ATRXImmunofluorescence(IF)results showed that shCon,shATRX1~#,shATRX2~#,and shATRX3~#HeLa cells all expressed GFP,suggesting that the lentivirus infection was successful,and ATRX protein was expressed in HeLa and shCon cells,but not in shATRX1~#,shATRX2~#,and shATRX3~#cells,suggesting that the three targeting sequences have better silencing efficiency;meanwhile,Western blotting results also confirmed this result.Based on this,HCT116 cells were infected with shATRX and shCon lentiviruses.Western blotting results showed that ATRX protein was expressed in shCon-HCT116 cells,but no or low expression in shATRX-HCT116 cells.These results proved that HCT116 cell models silenced ATRX were successfully obtained(p53+/+and p53-/-),and they can be used for subsequent experiments.2.The effects and molecular mechanisms of silencingATRX on radiation-induced HCT116 cell cycle arrestAt 24 and 48 h after 0,2 and 8 Gy irradiation,with both cells had G2/M phase arrest.When ATRX was silenced,both cells experienced S-phase delay,while G2/M arrest was decreased,and even HCT116(p53+/+)cells entered G0/G1 phase.Western blot results showed that after 2 and 8 Gy irradiation,the expression of p-ATM and p-Chk2 in the two types of cells increased,but after ATRX was silenced,both of the expressions were decreased as compared with them in control group.In addition,after ATRX silencing,the radiation-induced S-phase delay-related Cyclin A expression decreased,while the G2/M phase-associated proteins Cyclin B1 and CDK1were decreased in p53 knockout HCT116 cells and were increased in p53 wild-type HCT116 cells.And in HCT116 cells,the expressions of p-p53,p21 and Gadd45?were increased,suggesting that the cells had S-phase delay after ATRX silencing,and the radiation-induced G2/M phase arrest was decreased,but more cells in presence of p53 entered G0/G1 phase.ATRX is possible to control the corresponding cycle process by regulating p53 pathway.3.ATRX deletion enhances radiation-induced cell proliferation inhibitionThe results of CCK8 showed that after irradiation,the proliferation of HCT116cells slowed down,while the proliferation ability of cells silenced ATRX were decreased more significantly,and the proliferation ability of p53 wild-type HCT116cells were decreased more significantly(P<0.05).4.The effects of silent ATRX on the radiosensitivity of HCT16 cellsThe clonal formation was used to detect the changes in the surviving fractionof the cells.The results showed that after ATRX was silenced,the number of clones were decreased when the cells were irradiated with 0,2,4,6,and 8 Gy.After calculating the SF and fitting,the dose-survival curve was drawn,the curve slope of the HCT116cells were decreased,suggesting an increasing in radiosensitivity.5.ATRX deletion enhances radiation-induced DNA damageAfter 8 Gy irradiation,the fluorescence intensity of?H2AX and Rad51 in HCT116 cells were increased significantly from 3 to 6 h,and then began decrease,while the fluorescence intensity of?H2AX in shATRX-p53-/-cells was always at higher level,while the fluorescence intensity level of Rad51 was at lower level.Western Blot results showed that after irradiation,the expression of?H2AX and XRCC1 protein were increased,but the expression of Rad51 protein were decreased,and the expression of?H2AX protein in shATRX-p53-/-cells were increased most,suggesting that DNA damage is more serious in HCT116 cells silenced ATRX and knocked out p53.6.The effect of ATRX deletion on radiation-induced cell senescenceOn 0.5,1,2 and 4 days,the percentage of?-gal positive cells of p53 knockout HCT116 cells was not significantly different compared with them in the shCon and shATRX groups,but the percentage of?-gal positive cells of p53 wild-type HCT116cells increased significantly in the shCon group(P<0.05);and after 8 Gy irradiation,the percentage of?-gal positive cells in HCT116 cells were increased,but the percentage of?-gal positive cells in shATRX-p53+/+cells were increased less,while the percentage of?-gal positive cells were increased most in shATRX-p53-/-cells.HCT116 cells were stimulated with supernatants collected from 2 and 8 Gy irradiated cells,and cell supernatants from cells silenced ATRX had proliferation inhibiting effects.7.Loss of ATRX enhances radiation-induced apoptosisAfter 2 and 8 Gy irradiation,the apoptotic rate caused by radiation was increased in HCT116 cells silenced ATRX,but the apoptotic rate was higher in p53 wild-type HCT116 cells.At the same time,the expression levels of apoptosis-related protein PARP-1,cleaved caspase-9 and-3 were higher in HCT116 cells silenced ATRX.8.Relationship between ATRX/Daxx and p53The prediction of the STRING protein interaction found that there were interaction between MDM2,p53 and Daxx,and between Daxx and ATRX.It is speculated that ATRX may regulate p53.Western Blotting results showed that after ATRX was silenced,radiation-induced MDM2 and Daxx were decreased,and p-p53expression was increased,suggesting that ATRX negatively regulates p53.At 24 h after HCT116(p53+/+)cells were irradiated by 8 Gy,the whole proteins were extracted,and IP results confirmed that the interaction occurred between in ATRX with Daxx,but not with MDM2 and p53;but in Daxx with ATRX,MDM2 and p53.The results described as above suggested that ATRX negatively regulates p53indirectly through Daxx.Conclusions:1.All three sequences targeting-silenced ATRX can inhibit the expression of ATRX,and we constructed successfully HeLa cells and HCT116 cell models stably silenced ATRX.2.After silencing ATRX,HCT116 cells mainly suffer from S-phase delay,while ionizing radiation can induce G2/M phase arrest and enhance S-phase delay;when p53 is at presence,radiation could induce most HCT116 cells to enter G0/G1 phase.3.The radiation-induced proliferation in HCT116 cell silenced ATRX was more obvious,and it had effects on radiosensitization in HCT116 cells.4.After ATRX is silenced,radiation-induced DNA damage repair ability is reduced in HCT116 cells.5.Radiation can induce senescence of HCT116 cells,and after ATRX is silenced,senescence was decreased,and after p53 was knocked out,radiation-induced senescence were increased more significantly,it is possible that ATRX and p53synergistically inhibit radiation-induced cell senescence and have a positive effect on inhibition of cell proliferationcaused by radiation.6.Radiation can induce apoptosis of HCT116 cells,ATRX and p53 can cooperate to enhance the effects.7.WB and IP experiments confirmed that ATRX can participate in the negative feedback regulation of MDM2/p53 through Daxx.