靶向沉默Ku80对T-ALL细胞系Jurkat化疗敏感性影响的初步研究

Ku80 Inhibition Affects the Chemo the rapeutic Sensitivity of T-Acute Lymphoblastic Leukemia Cell Line Jurkat

目的:探讨靶向沉默Ku80对T-ALL细胞系Jurkat化疗敏感性的影响以及潜在机制。方法:通过RT-qPCR及Western blot技术检测6种不同血液肿瘤细胞系中Ku80基因的转录与表达水平;设计并构建Ku80特异性shRNA干扰质粒并转染T-ALL细胞系Jurkat后检测Ku80蛋白表达;CCK-8技术检测Ku80沉默后Jurkat细胞的增殖能力;软琼脂克隆形成技术、流式细胞术及Western blot技术分别检测Ku80沉默协同化疗药物依托泊苷(VP16)处理细胞4 h后Jurkat细胞的克隆形成能力、凋亡水平及DNA损伤蛋白γH2AX的表达水平。结果:在选取的6种不同血液肿瘤细胞系中,T-ALL细胞系Jurkat中Ku80的mRNA及蛋白水平均最高。构建的shRNA质粒成功靶向沉默了Jurkat细胞中Ku80的表达。Ku80靶向沉默后Jurkat细胞增殖能力明显下降(P<0.05);VP16孵育前后,Ku80靶向沉默均可显著降低Jurkat细胞克隆形成能力(P<0.01)、提高Jurkat细胞凋亡水平(P<0.01),并且γH2AX的表达水平显著提高(P<0.05)。结论:靶向沉默Ku80表达可增强T-ALL细胞系Jurkat对化疗药物VP16的敏感性,这或与其造成的DNA损伤积累水平增加有关。

Objective:To investigate the influence of Ku80 inhibition on the chemotherapeutic sensitivity of the T-acute lymphoblastic leukemia(T-ALL)cell line Jurkat,and to explore the potential mechanism.Methods:The transcription and expression level of Ku80 in 6 hematological malignant cell lines were detected by RT-qPCR and Western blot,respectively.The expression of Ku80 in Jurkat cells was detected by Western blot after transfection with the recombinant shKu80 lentiviral vector.The proliferation capacity of Jurkat cells was explored by CCK-8 after Ku80 inhibition.The colony formation ability,apoptosis,andγH2AX(a protein marker of DNA damage)expression in Jurkat cells were investigated after Ku80 silencing and co-treated with etoposide(VP16)for 4 hours through soft agar assay,flow cytometry and Western blot,respectively.Results:The mRNA level and protein expression of Ku80 were both highest in Jurkat among 6 hematological malignant cell lines.Ku80 expression was successfully down regulated in Jurkat cells after relative plasmid transfected.The proliferative ability of cells was significantly decreased after Ku80 inhibition(P<0.05).The colony formation capacity of Jurkat cells was obviously reduced and the cells apoptosis andγH2AX expression were increased after Ku80 inhibition,with or without VP16 incubation.Conclusion:Targeted silencing of Ku80 could enhance the sensitivity of VP16 in Jurkat cells,which might be associated with the elevated level of DNA damage accumulation.