CRISPR / Cas9 Gene Editing of QSOX1 in Breast Cancer Cells and Its Effect on the Proliferation and Invasion

doi:10.13523/j.cb.2007018

China Biotechnology

2020, Vol. 40

Issue (11): 1-9 DOI: 10.13523/j.cb.2007018

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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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

Received: 13 July 2020 Published: 11 December 2020

ZTFLH:

Q789

Corresponding Authors: Wen-yun ZHENG E-mail: zwy@ecust.edu.edu.cn

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	Xiu-juan HE
	Feng-zhi HU
	Qiu-li LIU
	Yu-ping LIU
	Ling ZHU
	Wen-yun ZHENG

Cite this article:

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.

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

Fig.1 Design of sgRNA targeting QSOX1 gene (a) Structure of pX459 plasmid (b) Recognition sequence of sgRNA targeting exons7 and 8 (c) Schematic diagram of expression vector pX459-QSOX1-sgRNA

Table 1 Oligonucleotide sequence of QSOX1-sgRNA

Fig.3 Schematic diagram of pX459-QSOX1-sgRNA mediated integration of exogenous expression cassettes into specific MCF-7 loci

Table 2 Primer sequences

Fig.2 Sequencing results of pX459-QSOX1-sgRNA plasmids

Fig.4 T7EN1 enzyme was employed to identify the mutation in the sgRNA sites

Fig.5 Generation and validation of stable cell line knock-out and knock-in QSOX1 gene (a), (b) QSOX1 protein expression in the selected monoclonal cells were confirmed by Western blot (c) Sequencing result of QSOX1 KO1, comparison of QSOX1 sequence in QSOX1 KO1 and the original QSOX1 sequence

Fig.6 Effect of QSOX1 gene knock-out and knock-in on the proliferation of MCF-7 cells (a) The proliferation curve (MTT) * P<0.05 (b) The results of colony formation assay

Fig.7 Effect of QSOX1 gene knock-out and knock-in on the migration and invasion of MCF-7 cells (a) Wound-healing assay (b) Transwell assay


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