CDC20 is a potential target gene to inhibit the tumorigenesis of MDCK cells

IF 1.5 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS
Zhenbin Liu , Mengyuan Pei , Geng Liu , Zhenyu Qiu , Siya Wang , Zilin Qiao , Jiamin Wang , Dongwu Jin , Jiayou Zhang , Kai Duan , Xuanxuan Nian , Zhongren Ma , Xiaoming Yang
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Abstract

MDCK is currently the main cell line used for influenza vaccine production in culture. Previous studies have reported that MDCK cells possess tumorigenic ability in nude mice. Although complete cell lysis can be ensured during vaccine production, host cell DNA released after cell lysis may still pose a risk for tumorigenesis. Greater caution is needed in the production of human vaccines; therefore, the use of gene editing to establish cells incapable of forming tumors may significantly improve the safety of influenza vaccines. Knowledge regarding the genes and molecular mechanisms that affect the tumorigenic ability of MDCK cells is crucial; however, our understanding remains superficial. Through monoclonal cell screening, we previously obtained a cell line, CL23, that possesses significantly reduced cell proliferation, migration, and invasion abilities, and tumor-bearing experiments in nude mice showed the absence of tumorigenic cells. With a view to exploring tumorigenesis-related genes in MDCK cells, DIA proteomics was used to compare the differences in protein expression between wild-type (M60) and non-tumorigenic (CL23) cells. Differentially expressed proteins were verified at the mRNA level by RT-qPCR, and a number of genes involved in cell tumorigenesis were preliminarily screened. Immunoblotting further confirmed that related protein expression was significantly reduced in non-tumorigenic cells. Inhibition of CDC20 expression by RNAi significantly reduced the proliferation and migration of MDCK cells and increased the proliferation of the influenza virus; therefore, CDC20 was preliminarily determined to be an effective target gene for the inhibition of cell tumorigenicity. These results contribute to a more comprehensive understanding of the mechanism underlying cell tumorigenesis and provide a basis for the establishment of target gene screening in genetically engineered non-tumorigenic MDCK cell lines.

CDC20是抑制MDCK细胞肿瘤发生的潜在靶基因
MDCK是目前用于流感疫苗培养生产的主要细胞系。先前的研究已经报道MDCK细胞在裸鼠中具有致瘤能力。尽管在疫苗生产过程中可以确保完全的细胞裂解,但细胞裂解后释放的宿主细胞DNA仍可能存在肿瘤发生的风险。在生产人类疫苗时需要更加谨慎;因此,利用基因编辑建立不能形成肿瘤的细胞可能会显著提高流感疫苗的安全性。关于影响MDCK细胞致瘤能力的基因和分子机制的知识是至关重要的;然而,我们的理解仍然是肤浅的。通过单克隆细胞筛选,我们先前获得了一种细胞系CL23,它具有显著降低的细胞增殖、迁移和侵袭能力,并且在裸鼠中的荷瘤实验显示不存在致瘤细胞。为了探索MDCK细胞中的肿瘤发生相关基因,使用DIA蛋白质组学来比较野生型(M60)和非肿瘤性(CL23)细胞之间蛋白质表达的差异。通过RT-qPCR在mRNA水平上验证了差异表达的蛋白质,并初步筛选了一些参与细胞肿瘤发生的基因。免疫印迹进一步证实,相关蛋白表达在非致瘤细胞中显著降低。RNAi对CDC20表达的抑制显著降低了MDCK细胞的增殖和迁移,并增加了流感病毒的增殖;因此,CDC20被初步确定为抑制细胞致瘤性的有效靶基因。这些结果有助于更全面地理解细胞肿瘤发生的机制,并为在基因工程非致瘤性MDCK细胞系中建立靶基因筛选提供基础。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Biologicals
Biologicals 生物-生化研究方法
CiteScore
3.70
自引率
0.00%
发文量
39
审稿时长
48 days
期刊介绍: Biologicals provides a modern and multidisciplinary international forum for news, debate, and original research on all aspects of biologicals used in human and veterinary medicine. The journal publishes original papers, reviews, and letters relevant to the development, production, quality control, and standardization of biological derived from both novel and established biotechnologies. Special issues are produced to reflect topics of particular international interest and concern.Three types of papers are welcome: original research reports, short papers, and review articles. The journal will also publish comments and letters to the editor, book reviews, meeting reports and information on regulatory issues.
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