摘要
目的:探讨GADD45A对骨肉瘤细胞的增殖和端粒调控功能。方法:应用siRNA和shRNA处理骨肉瘤细胞U2OS,观察骨肉瘤细胞增殖、端粒功能和端粒延长替代途径的变化。qPCR检测GADD45A敲低后mRNA水平。CCK-8实验和克隆形成实验检测骨肉瘤细胞增殖情况。细胞中期分裂相-荧光原位杂交实验检测端粒功能变化。免疫荧光-荧光原位杂交实验和C-circle实验检测端粒损伤情况和端粒延长替代途径相关表型。结果:qPCR结果表明,siRNA敲低GADD45A后mRNA水平降低(t=25.96,P<0.0001);shGADD45A-1和shGADD45A-2 GADD45A的mRNA水平降低(t=21.12、18.37,均P<0.0001)。对应的CCK-8(t=5.051、6.192、3.775、14.86、22.93、3.013、14.61、20.93,均P<0.05)和克隆形成实验(t=46.68、23.73、24.98,均P<0.0001)结果表明,敲低GADD45A抑制了骨肉瘤细胞增殖。细胞中期分裂相-荧光原位杂交实验结果显示,GADD45A缺失会加重端粒多信号(t=24.04、7.243、27.93,均P<0.01)和端粒信号缺失现象(t=8.222、16.61、6.781,均P<0.01)。免疫荧光-荧光原位杂交实验结果表明,siRNA敲低GADD45A后H2AX组蛋白变体和早幼粒细胞白血病体与端粒的共定位增加(t=19.16、10.65,均P<0.0001);shGADD45A-1、shGADD45A-2也证实了H2AX组蛋白变体和早幼粒细胞白血病体与端粒共定位的比例升高(t=14.71、24.03、16.69、18.10,均P<0.0001)。C-circle实验结果表明,siRNA敲低GADD45A以及shGADD45A-1和shGADD45A-2两细胞系中染色体外端粒DNA环C-circle水平升高(t=41.27、14.06、4.539,均P<0.05)。结论:GADD45A调控端粒延长替代途径、保护端粒功能并促进骨肉瘤细胞增殖。
Objective:To investigate the effect of GADD45A on the proliferation and telomere regulation of osteosarcoma cells.Methods:Osteosarcoma cell U2OS was treated with siRNA and shRNA to observe changes in cell proliferation,telomere function,and alternative lengthening of telomeres in osteosarcoma cells.The mRNA level of GADD45A knockdown was detected by qPCR.The proliferation of osteosarcoma cells was detected by CCK-8 assay and clone formation assay.Changes in telomere function were detected by metaphase phase-fluorescence in situ hybridization.Immunofluorescence fluorescence in situ hybridization and C-circle assay were used to detect telomere damage and phenotypes associated with alternative telomere lengthening pathways.Results:qPCR results showed that compared with NC group,mRNA level decreased after siRNA knockdown of GADD45A(t=25.96,P<0.0001).Compared with shscramble group,the mRNA level of GADD45A in shGADD45A-1 and shGADD45A-2 decreased(t=21.12,18.37,both P<0.0001).The corresponding CCK-8(t=5.051,6.192,3.775,14.86,22.93,3.013,14.61,20.93,all P<0.05)and colony formation assay(t=46.68,23.73,24.98,all P<0.0001)showed that deficiency of GADD45A inhibited the proliferation of osteosarcoma cells.The results of metaphase division phase fluorescence in situ hybridization showed that the loss of GADD45A could worsen the phenomenon of telomere multisignal(t=24.04,7.243,27.93,all P<0.01)and telomere signal loss(t=8.222,16.610,6.781,all P<0.01).The results of immunofluorescent-fluorescence in situ hybridization showed that the co-localization ofγ-H2AX and PML with telomere increased after siRNA treatment of GADD45A(t=19.16,10.65,both P<0.0001);shGADD45A-1 and shGADD45A-2 also confirmed the proportion ofγ-H2AX,PML and telomere co-localization increased(t=14.71,24.03,16.69,18.10,all P<0.0001).The results of C-circle assay showed that the level of C-circle in the outer telomere DNA circle increased among siGADD45A,shGADD45A-1 and shGADD45A-2 cell lines(t=41.27,14.06,4.539,all P<0.05).Conclusion:GADD45A regulates the alternative lengthening of telomere,protects telomere function and promotes the proliferation of osteosarcoma cells.
作者
韩鑫宇
李婷芳
王峰
HAN Xin-yu;LI Ting-fang;WANG Feng(Department of Genetics in Medicine,School of Basic Medical Science,Tianjin Medical University,Tianjin 300070,China)
出处
《天津医科大学学报》
2023年第3期265-271,共7页
Journal of Tianjin Medical University
