成簇规律间隔的短回文重复序列及其相关蛋白9敲除Ku80基因提高HEK293T细胞对阿霉素的敏感性

Ku80 gene knock out by clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 enhances the sensitivity of HEK293T cells to doxorubicin

目的利用成簇规律间隔的短回文重复序列及其相关蛋白9（CRISPR/Cas9）技术将293T细胞中的Ku80基因敲除，建立Ku80敲除的293T细胞株，并验证Ku80缺失对DNA损伤修复的影响以及对293T细胞阿霉素耐药性的影响。方法首先，设计识别针对靶位点的1对DNA Oligos，利用pSpCas9（BB）-2A-Puro （PX459）质粒构建表达导向RNA（sgRNA）载体。载体转染293T细胞。通过定点聚合酶链式反应（PCR）和T7E1内切酶酶切鉴定，确认所设计的sgRNA的有效性。进一步通过细胞有限稀释法和嘌呤霉素筛选，挑选单细胞克隆进行Western blot检测筛选出稳定敲除Ku80基因的293T细胞株。最后通过定点测序确认Ku80编码基因是否发生突变。使用阿霉素处理诱导DNA损伤，以磷酸化组蛋白H2AX（γ-H2AX）为指标检测DNA损伤，同时用细胞计数试剂盒（CCK-8）检测Ku80敲除的293T细胞对阿霉素的药物敏感性。结果成功构建Ku80基因敲除的293T细胞，用阿霉素处理诱导DNA损伤并经过修复之后，对照组和Ku80敲除组γ-H2AX染色的阳性率分别为（30.67±2.49）%和（88.00±1.63）%（P=0.000），表明Ku80敲除的293T细胞的DNA损伤修复功能显著受到抑制。同时Ku80敲除显著提高293T细胞对阿霉素的敏感性，阿霉素对Ku80敲除组细胞和对照组细胞的生长抑制率分别为（53.99±1.29）%和（32.42±1.20）%（P=0.000）。结论293T细胞中敲除Ku80抑制了DNA损伤的修复和提高了细胞对阿霉素的敏感性。

Objective293T cells with Ku80 knockout were constructed using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein9 （CRISPR/Cas9） technique, and the effect of Ku80 deletion on DNA damage repair and doxorubicin resistance of 293T cells was investigated.MethodsFirstly, a pair of DNA Oligos targeted Ku80 was designed and the single guide RNA （sgRNA） expression vectors were constructed using pSpCas9 （BB）-2A-Puro （PX459） vector. This vector was transfected into 293T cells. The effectiveness of the designed sgRNA was verified by site-directed Polymerase chain reaction （PCR） and T7E1 enzyme reaction. The 293T cell line was constructed using limited dilution method and puromycin screening. Single cell clones were selected and screened by Western blotting assay and sequencing. Furthermore, DNA damage was induced by doxorubicin treatment, and detected by DNA DSB marker phosphorylated Histone H2AX （γ-H2AX）. Cell counting kit-8 （CCK-8） assay was carried out to detect the sensitivity of the cells to doxorubicin.ResultsThe Ku80 gene knockout 293T cells were successfully constructed. After DNA damage induced by doxorubicin treatment and DNA repair, the positive rates of γ-H2AX staining in the control and Ku80 knockout groups were （30.67±2.49）% and （88.00±1.63）%, respectively （P=0.000）, indicating that the DNA damage repair ability was significantly inhibited in Ku80 knockout 293T cells. Ku80 knockout increases the sensitivity of 293T cells to doxorubicin. The growth inhibition rates of Ku80 knockout group and control group were （53.99±1.29）% and （32.42±1.20）%, respectively （P=0.000）.ConclusionThe knockout of Ku80 in 293T cells inhibited the DNA damage repair ability and increased the sensitivity of cells to doxorubicin.