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

您当前的位置：

首页 >

文章列表页 >

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

更新时间：2026-01-27

- Effects of Notch1 knockout by CRISPR-Cas9 on the proliferation and radiosensitivity of CNE2 cell line
- China Oncology Vol. 29, Issue 2, Pages: 111-118(2019)
- 作者机构：
  
  复旦大学附属肿瘤医院放疗科，复旦大学上海医学院肿瘤学系,上海,200032
- 作者简介：
- 基金信息：
- DOI：10.19401/j.cnki.1007-3639.2019.02.003
  CLC： R739.63
- Published Online：25 March 2019，
  
  Published：25 March 2019
- 稿件说明：
移动端阅览
汪宇洁，吕涛，王孝深. CRISPR-Cas9敲除Notch1对CNE2细胞增殖及辐射敏感性的影响[J]. 中国癌症杂志, 2019, 29(2): 111-118.

汪宇洁, 王孝深. Effects of Notch1 knockout by CRISPR-Cas9 on the proliferation and radiosensitivity of CNE2 cell line[J]. China Oncology, 2019, 29(2): 111-118.
汪宇洁，吕涛，王孝深. CRISPR-Cas9敲除Notch1对CNE2细胞增殖及辐射敏感性的影响[J]. 中国癌症杂志, 2019, 29(2): 111-118. DOI： 10.19401/j.cnki.1007-3639.2019.02.003.

汪宇洁, 王孝深. Effects of Notch1 knockout by CRISPR-Cas9 on the proliferation and radiosensitivity of CNE2 cell line[J]. China Oncology, 2019, 29(2): 111-118. DOI： 10.19401/j.cnki.1007-3639.2019.02.003.

摘要

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 Counting Kit-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（P0.05）；Notch1-KO组的D

0

、D

q

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，P0.001）和Ctrl组（1.182、2.516和0.819 Gy，P0.001）；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（P0.001）。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。

Abstract

Background and purpose: Notch signaling pathway plays a critical rol

e 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 (P0.001). Notch1 was not detected by Western blot and IF in Notch1-KO CNE2 cell line. Notch1 knockout inhibited cell proliferation of CNE2 cells (P0.05). The values of D

0

D

q

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) (P0.001) and those in Ctrl group (1.182

2.516 and 0.819 Gy) (P0.001). 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)%］ (P0.001) or the Ctrl group ［(3.70±0.03)%］(P0.001). 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.

关键词

Keywords

references

0

Views

2002

下载量

1

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Related Articles

The diagnostic value of zero echo time magnetic resonance imaging for skull base bone invasion in nasopharyngeal carcinoma

A comparative analysis of the long-term efficacy between partial hepatectomy and transcatheter arterial chemoembolization in patients with liver metastasis from nasopharyngeal carcinoma

Study on the mechanism of DDX6 promoting proliferation and migration of nasopharyngeal carcinoma cells by regulating stability of CKMT1A mRNA

Mechanism of circular RNA hsa_circ_0012779 expression in nasopharyngeal carcinoma and its influence on cell biological behavior

Application of generalized equivalent uniform dose optimization in the treatment of nasopharyngeal carcinoma with intensity-modulated radiotherapy

Related Author

Jiahao LIN

Meimei FENG

Kongqi LIN

Fengjie LIN

Yunbin CHEN

Yilin WANG

Lu WANG

Jiayan XIONG

Related Institution

Department of Radiation Oncology, Fujian Cancer Hospital, Clinical Oncology School of Fujian Medical University

Department of Radiology, Fujian Cancer Hospital, Clinical Oncology School of Fujian Medical University

Department of Liver Surgery, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University

Research Institute of Otolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University

Medical School of Nantong University

AI问答

⁰