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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (11): 1-9    DOI: 10.13523/j.cb.2007018
研究报告     
乳腺癌细胞QSOX1的CRISPR/Cas9基因编辑及其对增殖侵袭的影响研究*
何秀娟,胡凤枝,刘秋丽,刘玉萍,祝玲,郑文云()
华东理工大学药学院 上海市新药设计重点实验室 上海 200237
CRISPR / Cas9 Gene Editing of QSOX1 in Breast Cancer Cells and Its Effect on the Proliferation and Invasion
HE Xiu-juan,HU Feng-zhi,LIU Qiu-li,LIU Yu-ping,ZHU Ling,ZHENG Wen-yun()
School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
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摘要：

目的:乳腺癌细胞的恶性增殖和易于侵袭转移特性与其对患者的危害直接相关,因此,探究其产生的分子机制,对其有效防治具有重要意义。静息巯基氧化酶-1(QSOX1)是巯基氧化酶家族成员之一,有研究证明其对细胞内蛋白质折叠过程中二硫键形成及细胞外基质的形成发挥重要作用。由于QSOX1在乳腺癌和胰腺癌等多种癌细胞中过表达,将探索QSOX1对乳腺癌细胞过度增殖和侵袭转移方面的可能作用。方法:通过利用CRISPR/Cas9技术构建QSOX1基因敲除和敲入的乳腺癌细胞模型,检测分析QSOX1对乳腺癌细胞MCF-7的分裂增殖、侵袭迁移能力等方面的影响。结果:利用CRISPR/Cas9基因编辑技术成功构建了QSOX1基因敲除和敲入的乳腺癌MCF-7细胞株,其与对照野生型组细胞相比,QSOX1基因敲除株的增殖能力显著下降,癌细胞在体外的迁移和侵袭能力受到明显抑制;而QSOX1基因敲入株的增殖能力和体外迁移侵袭能力却明显有提高。结论:初步揭示了QSOX1在癌症发生与发展中的作用,为进一步阐明其作用的分子机制和设计靶向药物奠定了重要基础。
关键词： 静息巯基氧化酶-1(QSOX1);  乳腺癌MCF-7细胞;  增殖和侵袭能力;  CRISPR/Cas9基因编辑    
Abstract:

Objective: The malignant proliferation and easy invasion and metastasis of breast cancer cells are directly related to their harm to patients. Therefore, it is of great significance to explore the molecular mechanism of breast cancer cells for their effective prevention and treatment. QSOX1 is one of the members of thiol oxidase family. It has been proved that QSOX1 plays an important role in the formation of disulfide bond and extracellular matrix during protein folding. QSOX1 is over expressed in many kinds of cancer cells, including breast cancer and pancreatic cancer. The present study explored the possible role of QSOX1 in breast cancer cell proliferation, invasion and metastasis. Methods: By using CRISPR/Cas9 technology to construct QSOX1 gene knock-out and knock-in models of breast cancer cells, the effects of QSOX1 on the proliferation, invasion and migration of MCF-7 cells were analyzed. Results: The results showed that QSOX1 gene knock-out and knock-in MCF-7 cell lines were successfully constructed by CRISPR/Cas9 gene editing technology. Compared with WT cells, the proliferation ability of QSOX1 KO1 cells decreased significantly, the migration and invasion of cancer cells in vitro were significantly inhibited. However, the proliferation, migration and invasion capabilities of QSOX1 KI cells have been significantly improved. Conclusion: The study initially reveals the role of QSOX1 in the occurrence and development of cancer, and lays an important foundation for further elucidation of its molecular mechanism and design of targeted drugs.
Key words: QSOX1    MCF-7 cells    Proliferation and invasion    CRISPR /Cas9
收稿日期: 2020-07-13 出版日期: 2020-12-11
ZTFLH:  Q789  
基金资助: * 青年科学基金(81673345);国家自然科学基金面上项目(31670944);上海市科技创新项目(17431904600)
通讯作者: 郑文云     E-mail: zwy@ecust.edu.edu.cn
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引用本文:

何秀娟,胡凤枝,刘秋丽,刘玉萍,祝玲,郑文云. 乳腺癌细胞QSOX1的CRISPR/Cas9基因编辑及其对增殖侵袭的影响研究*[J]. 中国生物工程杂志, 2020, 40(11): 1-9.

HE Xiu-juan,HU Feng-zhi,LIU Qiu-li,LIU Yu-ping,ZHU Ling,ZHENG Wen-yun. CRISPR / Cas9 Gene Editing of QSOX1 in Breast Cancer Cells and Its Effect on the Proliferation and Invasion. China Biotechnology, 2020, 40(11): 1-9.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2007018    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I11/1

图1  靶向QSOX1基因的sgRNA的设计
sgRNA 序列(5'-3')
QSOX1-sgRNA1 F1: CACCGACCTGCCCACTTCTATCCGC
R1: AAACGCGGATAGAAGTGGGCAGGTC
QSOX1-sgRNA2 F2: CACCGGGAGCTATCTTGTTAGCAG
R2: AAACCTGCTAACAAGATAGCTCCC
QSOX1-sgRNA3 F3: CACCGTAAGCGGTATAGAAGGACC
R3: AAACGGTCCTTCTATACCGCTTAC
表1  QSOX1-sgRNA寡核苷酸序列
图3  pX459-QSOX1-sgRNA介导外源表达盒整合到MCF-7特定基因位点原理图
Prime Sequence(5'-3') The product of PCR
sgRNA1/2-F TCCAGAGCATGTGCAGTGAT 533bp
sgRNA1/2-R AAATGCCGCTCCCACACATT
SgRNA3-F GGCCTGGAGAGAAAACTCC 622bp
sgRNA3-R AGGCTGGAGGGTATCACCTAT
表2  引物序列
图2  pX459-QSOX1-sgRN重组质粒测序结果
图4  T7E1酶切鉴定突变体
图5  QSOX1基因敲除和敲入稳定细胞株的建立和鉴定
图6  QSOX1基因敲除和敲入对MCF-7细胞增殖的影响
图7  QSOX1基因敲除和敲入对MCF-7细胞迁移和侵袭能力的影响
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