羟基脲联合辐射对沉默ATRX后细胞周期及凋亡的影响

Effects of hydroxyurea combined with radiation on cell cycle and apoptosis of cells after silencing ATRX

目的:探讨羟基脲(HU)联合辐射对沉默α-地中海贫血/精神发育迟滞综合征X染色体相关蛋白(ATRX)后A549细胞周期和凋亡的影响,并阐明其可能的分子机制。方法:建立稳定沉默ATRX的A549细胞模型(shATRX-A549),荧光显微镜下观察细胞感染情况,采用Western blotting法检测沉默ATRX细胞中ATRX蛋白表达量验证细胞模型,以shNC-A549细胞作为阴性对照。实验分为对照组、HU组、辐射组(给予8 Gy X射线辐射)和HU+辐射组(给予HU+8 Gy X射线辐射)。流式细胞术检测各组不同细胞周期细胞百分率和细胞凋亡率,采用RNA测序(RNAseq)检测沉默ATRX后各组细胞中mRNA表达,采用Western blotting法检测各组细胞中细胞分裂周期因子(CDC25B)、细胞周期蛋白(Cyclin)B1和细胞周期蛋白依赖性激酶1(CDK1)蛋白表达量。结果:荧光显微下可见shNC-A549和shATRX-A549细胞表达绿色荧光蛋白(GFP);Western blotting法检测,与shNC-A549细胞比较,shATRX-A549细胞中ATRX蛋白表达量明显减少。流式细胞术检测,与对照组比较,HU组shNC-A549细胞中G_(0)/G_(1)期细胞百分率升高(P<0.05),S期和G_(2)/M期细胞百分率明显降低(P<0.05或P<0.01);辐射组shNC-A549细胞中G_(0)/G_(1)期和S期细胞百分率明显降低(P<0.01),G_(2)/M期细胞百分率明显升高(P<0.01);HU+辐射组shNC-A549细胞中G_(0)/G_(1)期细胞百分率明显降低(P<0.01),S期和G_(2)/M期细胞百分率明显升高(P<0.01);与对照组比较,HU组shATRX-A549细胞中G_(0)/G_(1)期细胞百分率升高(P<0.05),G_(2)/M期细胞百分率明显降低(P<0.01);辐射组shATRX-A549细胞中G_(2)/M期细胞百分率明显升高(P<0.01),G_(0)/G_(1)期和S期细胞百分率明显降低(P<0.01);HU+辐射组shATRX-A549细胞中G_(0)/G_(1)期细胞百分率明显降低(P<0.01),S期和G_(2)/M期细胞百分率明显升高(P<0.01);与shNC-A549细胞比较,辐射组shATRX-A549细胞中G_(0)/G_(1)期细胞百分率升高(P<0.05),G_(2)/M期细胞百分率降低(P<0.05);HU+辐射组shNC-A549细胞中S期细胞百分率升高(P<0.05)。与对照组比较,HU组、辐射组和HU+辐射组shNC-A549细胞和shATRX-A549细胞凋亡率明显升高(P<0.05或P<0.01)。与shNC-A549细胞比较,HU组和HU+辐射组shATRX-A549细胞凋亡率升高(P<0.05)。沉默ATRX后mRNA差异表达涉及c-Myc、Esp1、Cdc20、Plk1、CycA/B、Cip1和PCNA。Western blotting法检测,与对照组比较,HU组、辐射组和HU+辐射组shNC-A549和shATRX-A549细胞中CDC25B、Cyclin B1和CDK1蛋白表达量减少;与shNC-A549细胞比较,对照组和HU组shATRXA549细胞中Cyclin B1蛋白表达量略有减少,辐射组和HU+辐射组细胞中CDC25B、Cyclin B和CDK1蛋白表达量均增加。结论:HU和辐射均可导致沉默ATRX的A549细胞周期阻滞和细胞凋亡,其机制与CDC25B/Cyclin B/CDK1通路有关。

Objective:To investigate the effects of hydroxyurea(HU)combined with irradiation on the cycle cycle and apoptosis of the A549 cells after silencingα-thalassemia/mental retardation syndrome X stain-associated protein(ATRX),and to clarify the molecular mechanism.Methods:The A549 cell model(shATRX-A549)stable silencing ATRX was constructed,the infection situation of the cells was observed under fluorescence microscope,the expression of ATRX protein in the ATRX cells was detected by Western blotting method to verify the cell model,and the shNC-A549 cells were chosen as negative control.The experiment were divided into control group,HU group,radiation group(given 8 Gy X-ray radiation),and hydroxyurea+radiation group(given HU+8 Gy X-ray radiation).The percentages of cells at different cell cycle and apoptotic rates of the cells were detected by flow cytometry,and the expressions of mRNA in the cells after silencing ATRX were detected by RNA-sequencing(RNA-seq),and the expression amonts of the cell division cyclin 25B(CDC25B),Cyclin B1,and cyclin dependent kinase 1(CDK1)proteins in the cells in various groups were detected by Western blotting method.Results:The shNC-A549 cells and shATRX-A549 cells expressed green fluorescence protein(GFP)under fluorescence microscope;the Western blotting results showed that compared with shNC-A549 cells,the expression amount of ATRX protein in the shATRX-A549 cells was decreased.The flow cytometry results showed that compared with control group,the percentage of the shNC-A549 cells at G_(0)/G_(1) phase in HU group was increased(P<0.05),the percentages of the shNC-A549 cells at S phase and G_(2)/M phase were significantly decreased(P<0.05 or P<0.01),and the percentages of the shNC-A549 cells at G_(0)/G_(1) phase and S phase in radiation group were significantly decreased(P<0.01),while the percentage of the shNC-A549 cells at G_(2)/M phage was significantly increased(P<0.01),the percentage of the shNC-A549 cells at G_(0)/G_(1) phase in HU+radiation group was significantly decreased(P<0.01),and the percentages of the cells at S phase and G_(2)/M phase were significantly increased(P<0.01).Compared with control group,the percentage of the shATRX-A549 cells at G_(0)/G_(1) phase in HU group was increased(P<0.05),and the percentage of the shATRX-A549 cells at G_(2)/M phase was decreased(P<0.01);the percentage of the shATRX-A549 cells at G_(2)/M phase in radiation group was significantly increased(P<0.01),but the percentages of the shNC-A549 cells at G_(0)/G_(1) phase and S phase were significantly decreased(P<0.01),while the percentage of the shATRX-A549 cells at G_(0)/G_(1) phase in HU+radiation group was decreased(P<0.01),and the percentages of the shNC-A549 cells at S phase and G_(2)/M phase were significantly increased(P<0.05).Compared with the shNC-A549 cells,the percentage of the shATRX-A549 cells at G_(0)/G_(1) phase in radiation group was increased(P<0.05),the percentage of the shATRX-A549 cells at S phase in HU+radiation group was increased(P<0.05).Compared with control group,the apoptotic rates of the shNC-A549 cells and shATRX-A549 cells in HU group,radiation group,and HU+radiation group were significantly increased(P<0.05 or P<0.01).Compared with shNC-A549 cells,the apoptotic rates of the shATRX-A549 cells in HU group and HU+radiation group were increased(P<0.05).The differential expressions of mRNAs after silencing ATRX involved c-Myc,Esp1,Cdc20,Plk1,CycA/B,Cip1,and PCNA.The Western blotting results showed that compared with control group,the expression amounts of CDC25B,Cyclin B1,and CDK1 proteins in the shNC-A549 cells and shATRX-A549 cells in HU group,radation group and HU+radiation group were decreased;compared with the shNC-A549 cells,the expressions amounts of Cyclin B1 protein in the shATRX-A549 cells in control and HU groups were decreased,while the expression amounts of CDC25B,cyclin B1,and CDK1 proteins in the shATRX-A549 cells in radiation group and HU+radiation group were increased.Conclusion:HU and radiation can cause the cell cycle arrest and the apoptosis of the A549 cells after silencing ATRX,and its mechanism is related to the CDC25B/Cyclin B/CDK1 pathway.