抑制ATM表达对X线照射下肝癌细胞损伤修复的影响

Influence of siRNA targeting against ATM on DNA damage repair for hepatoma cells after X irradiation

目的探讨抑制毛细血管扩张-共济失调突变基因(ATM)表达对人肝癌细胞株HepG2在X线照射下细胞损伤修复的影响。方法设计3条沉默ATM的小分子干扰RNA(siRNA)的序列,用脂质体法转染HepG2细胞,筛选出有效的浓度及作用时间,分别利用实时定量PCR和蛋白质印迹法检测ATM mRNA、蛋白表达水平,MTT法检测转染后细胞增殖,流式细胞术检测X线照射后HepG2细胞周期、凋亡等生物学功能方面指标的变化。结果筛选出沉默ATM基因的siRNA有效序列。qRT-PCR结果显示第3条siRNA抑制率最高,可达72%,转染后48 h的ATM蛋白表达抑制78%。转染siRNAATM后HepG2细胞的增殖能力无明显变化,HepG2ATM组在X线照射后G2/M期细胞明显增多,S期细胞明显减少;X线照射后HepG2ATM组早期凋亡百分比是阴性对照组的2倍。结论抑制ATM表达可显著抑制肝癌细胞对辐射损伤的修复,为后期研究ATM在肝癌治疗中的作用机制提供了实验基础。

Objective To study the influence of RNA interference targeting against ataxia-telangiectasia mutated（ATM） gene on DNA damage repair for human hepatoma cells HepG2 after X irradiation.Methods According to the genetic information,three siRNA sequences of ATM were designed,then transfected into the hepatoma cell by liposome,and chosed the effective concentration and time.The expression level of ATM mRNA and protein was detected by qRT-PCR and Western blotting respectively.The cell proliferation was assessed by MTT assay and the changes in cell cycle and apoptosis were evaluated by flow cytometry.Results The valid siRNA sequence which could depress ATM gene was boltinged.qRT-PCR demonstrated that the best inhibitory effect on ATM mRNA expression in HepG2 was 72% in the third siRNA sequence.Western blot demonstrated the inhibition ratio on protein expression was 78% in 48 h after transfection.The proliferation of HepG2 cells was not marked changing by siRNAATM.The number of cells at G2/M stage in HepG2ATM group was obviously increased,yet the number of cells at S stage was manifestly decreased after X irradiation.FCM showed the period apoptosis ratio in HepG2ATM group was 2 folds than that of the negative control group.Conclusion The RNA interference targeting against ATM gene significantly inhibites DNA repair response of hepatoma cells after radiation damage.It may be helpful to provide solid foundation for studying the function of ATM in the therapy of liver cancer.