Effects And Mechanisms Of Silencing ATRX On Mitotic Catastrophe Induced By Radiation In Tumor Cells

Nowadays,cancer is still one of the serious diseases to threaten the public health.According to the data of The International Cancer Agency in 2020,China is the first country with new tumors,which seriously affects people’s health and quality of life.The treatment of cancer has always been a topic of close concern in the society.Radiotherapy is an important means of tumor treatment.It can cause cell DNA damage by direct or indirect effects,stop cell cycle process,activate cell death pathway and finally kill tumor cells.Mitotic catastrophe is one way to remove damaged cells induced by DNA damage after exogenous stimulation.According to its ability to maintain the stability of the genome,inducing mitotic catastrophe in tumor cells is expected to achieve the purpose of eliminating tumor cells.α-thalassemia/mental retardation syndrome X-linked（ATRX）has the biological functions of DNA damage repair and the chromosome telomere stability.In this study,tumor cell models stably silenced ATRX were established to explore the effects and relative molecular mechanisms of ionizing radiation on tumor cell proliferation,cell cycle process,mitotic catastrophe,apoptosis and senescence,so as to provide a new explanation for the killing effect of tumor cells caused by radiation and a new molecular target for tumor therapy.Objective:To explore the effect and molecular mechanism of targeted silencing ATRX on tumor cell proliferation,cell cycle process,mitotic catastrophe,apoptosis and senescence induced by ionizing radiation,so as to provide a new explanation for the killing effect of radiation on tumor cells.Methods:1.Three sh RNA sequences（sh ATRX）targeting ATRX were designed and cloned into lentiviral vector p GIPz,breast cancer cells MDA-MB-231（mt-p53）,colon cancer cell HCT116（wt-p53）and HCT116（null-p53）were infected after packaging lentivirus using 293T cells,non target sequence was used for control（sh Con）.Western blot（WB）was used to detect the protein expression of ATRX.The expression of GFP was observed under fluorescence microscope to determine the infection efficiency.Tell lines with good silencing efficiency were selected for follow-up experiment,named NC and ATRX^low..2.X-RAD 320i X deep irradiator is used for irradiation.The irradiation conditions:voltage 180 k V,current 20 m A,target distance 70 cm,dose rate 1.0 Gy/min.3.5-ethynyl-2’-deoxyuridine（EdU）staining was used to detect the proliferation changes of MDA-MB-231（mt-p53）,HCT116（wt-p53）and HCT116（null-p53）cells24 hours after 0,4 and 8 Gy irradiation.4.The changes of cell cycle and polyploidy of MDA-MB-231（mt-p53）,HCT116（wt-p53）and HCT116（null-p53）cells of NC and ATRX^low were detected by flow cytometry using PI staining at 24 hours after 0,4 and 8 Gy irradiation.And the expression of cycle related proteins was detected by WB.5.The nuclear and spindle morphology of MDA-MB-231（mt-p53）,HCT116（wt-p53）and HCT116（null-p53）cells of NC and ATRX^low were observed by immunofluorescence at 24 hours after 0,4 and 8 Gy irradiation.The expressions of mitotic related proteins Cyclin B1,CDK1,Cyclin D1,CDK4,p53 and MDM2 were detected by WB.6.The changes of apoptosis rate of MDA-MB-231（mt-p53）,HCT116（wt-p53）and HCT116（null-p53）of NC and ATRX^low were detected by flow cytometry using Annexin V-PE/7-ADD apoptosis kit at 24 hours after 0,4 and 8 Gy irradiation.And the expression of apoptosis related proteins caspase 3 and cleaved-caspase 3 was detected by WB.7.The changes of the senescence positive rate of MDA-MB-231（mt-p53）,HCT116（wt-p53）and HCT116（null-p53）of NC and ATRX^low were detected byβ-Galactosidase cell senescence detection kit at 24 hours after 0,4 and 8 Gy irradiation.8.SPSS 24.0 was used for statistical analysis.The percentage of cell proliferation,the percentage of cell cycle distribution,the percentage of cell apoptosis and the percentage of aging cells are all expressed inx±s after the normality test,they all conform to the normal distribution.The analysis of variance is used for the comparison of sample mean among multiple groups,and the independent sample t-test is used for the comparison between two groups.P<0.05 indicates that the difference is statistically significant;Use graphpad prism 8 software to draw the result diagram.Results:1.Construction of cell models silenced ATRXBased on the targeted silencing ATRX sequence,293T cells were used to harvest non target control（sh Con）and targeted silencing ATRX（sh ATRX）lentiviruses,after0.45μm filtration,MDA-MB-231（mt-p53）,HCT116（wt-p53）and HCT116（null-p53）cells were infected respectively.WB results showed that ATRX was highly expressed in parent cells and sh Con cells of the three cells,while the expression of sh ATRX1,sh ATRX2 and sh ATRX3 cells was decreased in various degrees,especially in sh ATRX1 cells,suggesting that the silencing efficiency was the highest.The expression of GFP in sh Con and sh ATRX1 cells was observed under fluorescence microscope.Under the microscope,higher GFP protein expression was observed,indicating higher infection efficiency.The above results indicate that the cell model of targeted silencing ATRX has been successfully established in this study,and sh Con and sh ATRX1 cells were selected for follow-up research,named NC and ATRX^low respectively.2.Silencing ATRX enhanced radiation-induced inhibition of cell proliferationThe positive cells were detected after EdU staining.The results showed that the percentage of EdU positive cells in MDA-MB-231（mt-p53）,HCT116（wt-p53）and HCT116（null-p53）decreased at 24 hours after 4 and 8 Gy irradiation compared with 0Gy（P<0.05）,and when the dose was higher,the EdU positive cells was fewer.Compared with NC cells,the EdU positive rate of ATRX^low cells was further reduced（P<0.05）,and the inhibition effects of HCT116（null-p53）cells of ATRX^low was more obvious than the other two cells3.Effects and mechanisms of silencing ATRX on cell cycle progression after radiation（1）Silencing ATRX enhanced cell cycle arrest after radiationThe flow cytometry results showed that compared with 0 Gy,the proportion of cells in G₂/M phase increased at 24 hours after 4 and 8 Gy irradiation（P<0.05）,and G₂/M phase arrest occurred,with the increase of dose,G₂/M phase arrest was more obvious（P<0.05）.Moreover,in HCT116（wt-p53）cells,compared with NC,after 4and 8 Gy irradiation,the proportion of cells in G₀/G₁ phase and S phase in ATRX^lowcells increased（P<0.05）,G₁ phase arrest and S phase delay occurred,but the similar changes were not found in MDA-MB-231（mt-p53）cells and HCT116（null-p53）cells.Author speculates that the cycle arrest of HCT116（wt-p53）cells caused by silencing ATRX is different from the other two cells by regulating the expression of p53.（2）Silencing ATRX affected the expression of cycle proteins after radiationWB results showed that compared with 0 Gy,the expression of G₂/M phase relative protein Cyclin B1 and CDK1 in MDA-MB-231（mt-p53）and HCT116（null-p53）cells were increased after 4 and 8 Gy irradiation,especially higher after 4 Gy irradiation,and the expression of G₀/G₁ phase related protein Cyclin D1 was decreased after irradiation,but CDK4 did not change significantly.Under the same dose,compared with NC cells,the expressions of Cyclin B1 and CDK1 were decreased,Cyclin D1 was increased,and the change of CDK4 was not obvious in MDA-MB-231（mt-p53）and HCT116（null-p53）of ATRX^low cells.In HCT116（wt-p53）cells,compared with 0 Gy,the expression of Cyclin B1,CDK1 and Cyclin D1 were decreased slightly after 4 and 8 Gy irradiation,but the change of CDK4 was not obvious.And compared with NC cells,the expression of Cyclin B1 and CDK1 in ATRX^low cells were decreased,Cyclin D1 was decreased,but the change of CDK4 was not obvious.（3）Silencing ATRX enhanced the expression of p53 proteinUsing the bioinformatics website Gene MANIA,we found that there was an interaction between ATRX and Daxx,MDM2 and Daxx,MDM2 and p53.Author speculated that there might be a regulatory relationship between ATRX and p53.WB results showed that the expression of ATRX,Daxx and MDM2 were increased after radiation,the expression of p53 in MDA-MB-231（mt-p53）and HCT116（wt-p53）cells was also increased.After silencing ATRX,the expression of Daxx and MDM2 were decreased,but p53 was increased further in MDA-MB-231（mt-p53）and HCT116（wt-p53）cells.4.Silencing ATRX enhanced radiation induced mitotic catastrophe（1）Silencing ATRX induced abnormal karyokinesis after radiationThe results showed that after irradiation,micronucleus appeared in MDA-MB-231（mt-p53）cells,and the same phenomenon was observed in HCT116（null-p53）of ATRX^low cells,but there was no obvious abnormal nuclear morphology in HCT116（wt-p53）cells.（2）Silencing ATRX affected the production of polyploid after radiationThe results of flow cytometry showed that after 4 and 8 Gy irradiation,the percentage of hexaploidy and octaploid cells of MDA-MB-231（mt-p53）and HCT116（wt-p53）cells were increased,and the increase of ATRX^low cells was more obvious（P<0.05）.In HCT116（null-p53）cells,the percentage of octaploid cells was increased after irradiation,and the increase was more obvious in ATRX^low group（P<0.05）.（3）Effect of silencing ATRX on spindle morphology of irradiated cellsThe spindle morphology was observed by immunofluorescence method,α-Tubulin,main components of spindle was stained,the results showed that in MDA-MB-231（mt-p53）cells,after 4 and 8 Gy irradiation,the spindle was abnormal and multipolar spindle appeared,and multipolar spindles appeared in MDA-MB-231（mt-p53）and HCT116（null-p53）cells of ATRX^low after 4 and 8 Gy irradiation,however,in HCT116（wt-p53）cells,there are no or rare abnormal cell spindles.（4）Silencing ATRX inhibited the protein expression of Aurora B after radiationThe chromosome chaperone passenger complex（CPC）is very important for gene transmission during mitosis.Aurora B and CENPA are important components of the complex,especially Aurora B,which plays a key role in the formation and localization of centrosome.The expression of Aurora B and its important substrates p H3 and CENPA were detected by WB,the results showed that compared with 0 Gy,the expression of Aurora B,CENPA and p H3 were increased after 4 Gy irradiation and decreased after 8 Gy irradiation.At the same dose,the expression of Aurora B,CENPA and p H3 in ATRX^low cells was lower than NC cells.（5）Silencing ATRX affected the function of SAC after radiationSAC is the last checkpoint in cell mitosis and plays an important monitoring role in the process of mitosis.The dysfunction of SAC is directly related to MC.WB results showed that compared with 0 Gy,the expression of CDC20 and Bub1b were increased after 4 Gy irradiation,decreased after 8 Gy irradiation in three cells,and the expression of MAD2 did not change significantly after irradiation.Under the same dose irradiation,the expression of CDC20,Bub1b and MAD2 proteins were decreased in ATRX^low cells compared with NC cells.5.Silencing ATRX enhanced radiation-induced apoptosisThe results of flow cytometry showed that compared with 0 Gy,the apoptosis rate of MDA-MB-231（mt-p53）,HCT116（wt-p53）and HCT116（null-p53）cells was increased significantly after 4 and 8 Gy irradiation（P<0.05）.Under the same dose irradiation,compared with NC cells,the apoptosis rate in MDA-MB-231（mt-p53）and HCT116（wt-p53）cells of ATRX^low was higher（P<0.05）,while in HCT116（null-p53）cell of ATRX^low had no significant change.WB results showed that compared with 0Gy,the expression of apoptosis related protein caspase 3 was decreased and cleaved-caspase 3 was increased in the three cells after irradiation,and the related changes were more obvious in ATRX^low cells compared with NC cells.6.Effect of silencing ATRX on radiation induced cell senescenceβ-Galactosidase detection kit was used to detect the senescence cells after irradiation.The results showed that in HCT116（wt-p53）and HCT116（null-p53）cells,compared with 0 Gy,the number ofβ-galactosidase positive cells began to increase at1 d（P<0.05）,indicating that the cells were aging,and when the dose was higher,the senescence percentage was greater,and reached the peak at 4 d post-irradiation.Compared with NC cells,the senescence percentage of ATRX^low cells was significantly lower（P<0.05）.However,in MDA-MB-231（mt-p53）cells,after 4 and 8 Gy irradiation,the senescence percentage was not significantly different from that of 0 Gy.And there was no significant difference between NC and ATRX^low cells.Conclusions:1.The cell models of MDA-MB-231（mt-p53）,HCT116（wt-p53）and HCT116（null-p53）silenced ATRX were successfully established.Silencing ATRX can enhance the inhibition of cell proliferation induced by radiation,and the inhibition is more obvious in HCT116（null-p53）cells;2.Radiation can induce G₂/M phase arrest in three kinds of tumor cells,while silencing ATRX enhanced G₀/G₁ phase arrest and S phase delay in p53 wild cells after radiation,it may be related to the negative regulation of p53 by ATRX loss through Daxx/MDM/p53 pathway;3.Radiation induces mitotic catastrophe in tumor cells by disturbing the function of SAC,and silencing ATRX can increase the mitotic catastrophe of irradiated cells;4.Silencing ATRX enhances cell apoptosis after radiation and reduces radiation-induced cell senescence,especially the cell senescence of HCT116（wt-p53）and HCT116（null-p53）cells,which may be related to the composition of senescence related heterochromatic lesions by ATRX.