Chk1/2反义寡核苷酸对顺铂诱导的A549细胞生物学行为的影响

Effects of Antisense Oligonucleotide Targeting Chk1/2 on Apoptosis of A549 Cells Induced by DDP

目的观察顺铂作用下A549细胞的细胞周期变化规律和Chk1、Chk2(Chk1/2)反义寡核苷酸(AsODN)对顺铂(DDP)诱导的A549细胞生物学行为的影响。方法流式细胞术SubG1法检测DDP作用下A549细胞周期和凋亡的动力学变化;蛋白免疫印迹法(Westernblot)检测转染Chk1/2AsODN后Chk1/2蛋白的表达;流式细胞仪AnnexinⅤ-FITC法和SubG1法检测转染Chk1/2AsODN后DDP作用下的细胞周期和凋亡变化。结果10μmol/LDDP作用12h后A549细胞出现明显的S期阻滞,Chk1/2AsODN对A549细胞中Chk1/2蛋白表达有明显抑制作用,转染Chk1/2AsODN可显著增加DDP诱导下A549细胞的凋亡率约200%～300%,而联合转染Chk1/2AsODN与单转染相比,凋亡无明显增加(P>0.05)。转染Chk1/2AsODN引起化疗增敏的机制是通过解除S期阻滞这种肿瘤细胞的自我保护机制实现的。结论Chk1/2可作为肺癌化疗药物增敏治疗的有效靶点,灭活Chk1/2基因可以显著增强肿瘤细胞化疗的敏感性。

Objective To investigate the change of cell cycle induced by cisplatin （DDP） and the effects of antisense oligonueleotide （AsODN） targeting Chk1/2 on apoptosis induced by DDP in A549 cells. Methods The Chk1/2 protein expression was detected by Western blot after transfection of AsODN targeting Chk1/2 by lipofectamine into A549 cells. Apoptosis of A549 cells induced by DDP was assayed by flow cytometry after transfection of Chk1/2 AsODN. Results Asynchronized A549 cells were treated with different doses of DDP to activate S checkpoints. Transfection with Chk1/2 AsODN could inhibit the expression of Chk1/2 at different levels. Either Chk1 or Chk2-speeific AsODN consistently enhanced DNA damage-induced apoptosis by 200%-300% as compared with corresponding sODN controls （P〈0. 05）. Unexpectedly, combined use of Chk1- and Chk2-specific AsODN did not produce synergistic effects as compared to treatment with Chk1-or Chk2-specific AsODN alone （P〉0.05）. The reason for chemosensitivity by transfection of Chk1/2 AsODN may be that the self-control of tumor cells was destroyed by relieving cell cycle S phase arrest. Conclusion Chk1/2 may be regarded as targets of chemotherapy for lung cancer, and blocking Chk1/2 gene could obviously sensitize tumor ceils to chemotherapy in lung cancer.