{"title":"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","authors":"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","doi":"10.1016/j.bej.2024.109553","DOIUrl":null,"url":null,"abstract":"<div><div>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 IC<sub>50</sub> 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.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"213 ","pages":"Article 109553"},"PeriodicalIF":3.7000,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"CRISPR/Cas9-mediated knockout of DYRK1B in triple-negative breast cancer cells: implications for cell proliferation, apoptosis, and therapeutic sensitivity\",\"authors\":\"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\",\"doi\":\"10.1016/j.bej.2024.109553\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>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 IC<sub>50</sub> 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.</div></div>\",\"PeriodicalId\":8766,\"journal\":{\"name\":\"Biochemical Engineering Journal\",\"volume\":\"213 \",\"pages\":\"Article 109553\"},\"PeriodicalIF\":3.7000,\"publicationDate\":\"2024-10-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biochemical Engineering Journal\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1369703X24003401\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemical Engineering Journal","FirstCategoryId":"5","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1369703X24003401","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
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.
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.