CRISPR-Cas9敲除Notch1对CNE2细胞增殖及辐射敏感性的影响

Effects of Notch1 knockout by CRISPR-Cas9 on the proliferation and radiosensitivity of CNE2 cell line

背景与目的:Notch信号转导通路对鼻咽癌的发生、发展及维持肿瘤干细胞的自我更新等具有重要作用,既往研究方法难以使Notch通路中特定基因功能完全丧失。本研究旨在利用CRISPR-Cas9基因编辑技术构建Notch1基因敲除的CNE2鼻咽癌细胞系,并观察Notch1敲除对CNE2细胞增殖、辐射敏感性的影响。方法:采用蛋白质印迹法(Western blot)检测人鼻咽癌细胞CNE2及鼻咽正常上皮细胞NP69中Notch1表达水平;利用sgRNA在线设计工具,针对Notch1设计sgRNA;利用PX459质粒构建含sgRNA的敲除载体PX459-Notch1-sgRNA;将质粒瞬时转染CNE2细胞,经过药物筛选、克隆化培养、Western blot检测、免疫荧光染色验证得到Notch1敲除的CNE2鼻咽癌细胞系;采用细胞计数试剂盒(Cell CountingKit-8,CCK-8)检测方法、克隆形成实验检测Notch1敲除组(Notch-KO)与未转染亲本组(Parental)、转染PX459空载体水平较的对照组(Ctrl)的增殖活性及辐射敏感性,并进一步采用流式细胞术检测侧群细胞比例。结果:与NP69细胞相比,CNE2细胞中Notch1蛋白水平较高;Western blot检测、免疫荧光染色验证Notch1基因被完全敲除。Notch1基因敲除可抑制细胞增殖活性(P<0.05);Notch1-KO组的D0、Dq和SF2值分别为1.160、1.881和0.630 Gy,低于Parental(1.176、2.533和0.824 Gy,P<0.001)和Ctrl组(1.182、2.516和0.819 Gy,P<0.001);Notch1-KO中侧群细胞比例[(1.13±0.01)%]较Parental[(3.81±0.03)%]、Ctrl[(3.70±0.03)%]明显下降(P<0.001)。结论:运用CRISPRCas9技术可成功敲除CNE2细胞中的Notch1基因,Notch1基因敲除导致CNE2细胞增殖能力下降、辐射敏感性增加、侧群细胞比例降低。

Background and purpose: Notch signaling pathway plays a critical role in the development of nasopharyngeal carcinoma(NPC) and self-renewal of cancer stem cells(CSC). Complete loss of biological functions of specific gene is difficult to achieve by previous methods in Notch pathway. This study aimed to establish Notch1 knockout NPC CNE2 cell line using CRISPRCas9 gene editing technology and to explore the effects of Notch1 on the proliferation and radiosensitivity of CNE2. Methods:Western blot was applied to determine the protein level of Notch1 in NPC cell line CNE2 and normal nasopharyngeal epithelial cell line NP69. SgRNA online design tool was used to design sgRNA for Notch1. PX459 plasmid was utilized to construct knockout vector containing the sgRNA named PX459-Notch1-sgRNA. Notch1 knockout CNE2 cell line was constructed through transfection,drug screening and cloning, followed by verification via Western blot and immunofluorescence(IF). Then Cell Count Kit-8(CCK-8), colony formation assay and flow cytometry were used respectively to detect cell proliferation, radiation sensitivity and sidepopulation cell ratio in group with Notch1 knockout(Notch1-KO), group without transfection(Parental) and group only transfected with PX459(Ctrl). Results: Notch1 protein level was higher in CNE2 than NP69(P<0.001). Notch1 was not detected by Western blot and IF in Notch1-KO CNE2 cell line. Notch1 knockout inhibited cell proliferation of CNE2 cells(P<0.05). The values of D0,Dq and SF2 in Notch1-KO group were 1.160, 1.881 and 0.630 Gy, respectively, obviously lower than those in Parental group(1.176,2.533 and 0.824 Gy)(P<0.001) and those in Ctrl group(1.182, 2.516 and 0.819 Gy)(P<0.001). There was significant difference in side population cell ratio between the Notch1-KO [(1.13±0.01)%] group and the Parental group[(3.81±0.03)%](P<0.001)or the Ctrl group [(3.70±0.03)%](P<0.001). Conclusion: CRISPR-Cas9 technology can successfully knockout Notch1 gene of CNE2 cell line. Notch1 knockout results in suppression of CNE2 cell proliferation, radiation sensitization and down-regulation of side population cell ratio.