CRISPR/Cas9 介导的三阴性乳腺癌细胞 DYRK1B 基因敲除:对细胞增殖、凋亡和治疗敏感性的影响

IF 3.7 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biochemical Engineering Journal Pub Date : 2024-10-29 DOI:10.1016/j.bej.2024.109553
Asrin Rashidi , Ernst-Martin Füchtbauer , Zakaria Vahabzadeh , Farzad Soleimani , Karim Rahimi , Bahram Nikkhoo , Shohreh Fakhari , Mohammad Bagher Khadem Erfan , Asaad Azarnezhad , Arash Pooladi , Fariborz Soheili , Fardin Fathi
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引用次数: 0

摘要

乳腺癌是全球妇女最常见的癌症,其中三阴性亚型(TNBC)预后较差,治疗方案有限。DYRK1B 是一种双重特异性激酶,可调节细胞周期和静止状态。虽然DYRK1B在多种癌症中的作用已被证实,但它在TNBC中的作用仍不清楚。在本研究中,我们使用 CRISPR/Cas9 技术在 TNBC 模型 MDA-MB-231 细胞中删除了 DYRK1B,并研究了它对细胞增殖、凋亡、侵袭、迁移、血管生成和对紫杉醇反应的影响。通过 PCR、实时 qPCR 和 Sanger 测序证实了 DYRK1B 基因敲除(KO)。KO细胞在细胞增殖、集落形成、侵袭和迁移方面均表现出明显的下降。此外,与 MDA-MB-231 野生型(WT)细胞相比,与细胞周期、血管生成和细胞运动相关的几个基因(如 CCND1、MCM2、PCNA、CDKN1B、HIF1A、VEGFA 和 WASF3)的 mRNA 表达水平也发生了变化。免疫细胞化学评估细胞增殖标志物 Ki67 表达的结果表明,DYRK1B 基因敲除细胞的 Ki67 表达明显低于 WT 细胞。此外,KO 细胞表现出更强的凋亡能力和对接触抑制的敏感性。此外,KO 细胞中紫杉醇的 IC50 值明显降低。这些结果表明,DYRK1B在TNBC细胞的存活和侵袭过程中起着重要作用,可能成为治疗这种疾病的新靶点。
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CRISPR/Cas9-mediated knockout of DYRK1B in triple-negative breast cancer cells: implications for cell proliferation, apoptosis, and therapeutic sensitivity
Breast cancer is the most common cancer among women worldwide, with the triple-negative subtype (TNBC) having a poor prognosis and limited treatment options. DYRK1B is a dual-specificity kinase that regulates the cell cycle and quiescence. While its role in several cancers has been characterized, its role in TNBC remains unknown. In this study, we used CRISPR/Cas9 to delete DYRK1B in MDA-MB-231 cells, a model of TNBC and investigated its effects on cell proliferation, apoptosis, invasion, migration, angiogenesis, and response to Paclitaxel. The DYRK1B knockout (KO) was confirmed by PCR, Real-time qPCR, and Sanger sequencing. KO cells showed a significant reduction in cell proliferation, colony formation, invasion, and migration. Additionally, there were alterations in mRNA expression levels of several genes related to the cell cycle, angiogenesis, and cell motility, such as CCND1, MCM2, PCNA, CDKN1B, HIF1A, VEGFA, and WASF3, compared to MDA-MB-231 wild type (WT) cells. Immunocytochemistry results assessing Ki67 expression, a marker of cell proliferation, indicated that DYRK1B knockout cells had significantly lower Ki67 expression than WT cells. Furthermore, KO cells exhibited increased apoptosis and sensitivity to contact inhibition. Additionally, the IC50 for Paclitaxel was significantly decreased in KO cells. These results suggest that DYRK1B plays an important role in the survival and invasion of TNBC cells and might be a potential candidate as a new therapeutic target for this disease.
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来源期刊
Biochemical Engineering Journal
Biochemical Engineering Journal 工程技术-工程:化工
CiteScore
7.10
自引率
5.10%
发文量
380
审稿时长
34 days
期刊介绍: The Biochemical Engineering Journal aims to promote progress in the crucial chemical engineering aspects of the development of biological processes associated with everything from raw materials preparation to product recovery relevant to industries as diverse as medical/healthcare, industrial biotechnology, and environmental biotechnology. The Journal welcomes full length original research papers, short communications, and review papers* in the following research fields: Biocatalysis (enzyme or microbial) and biotransformations, including immobilized biocatalyst preparation and kinetics Biosensors and Biodevices including biofabrication and novel fuel cell development Bioseparations including scale-up and protein refolding/renaturation Environmental Bioengineering including bioconversion, bioremediation, and microbial fuel cells Bioreactor Systems including characterization, optimization and scale-up Bioresources and Biorefinery Engineering including biomass conversion, biofuels, bioenergy, and optimization Industrial Biotechnology including specialty chemicals, platform chemicals and neutraceuticals Biomaterials and Tissue Engineering including bioartificial organs, cell encapsulation, and controlled release Cell Culture Engineering (plant, animal or insect cells) including viral vectors, monoclonal antibodies, recombinant proteins, vaccines, and secondary metabolites Cell Therapies and Stem Cells including pluripotent, mesenchymal and hematopoietic stem cells; immunotherapies; tissue-specific differentiation; and cryopreservation Metabolic Engineering, Systems and Synthetic Biology including OMICS, bioinformatics, in silico biology, and metabolic flux analysis Protein Engineering including enzyme engineering and directed evolution.
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