Knockout Of BIRC5 Gene By CRISPR/Cas9 Induces Apoptosis And Inhibits Cell Proliferation In Leukemic Cell Lines, HL60 And KG1
Affiliations
Affiliations
- 1 Cancer and Immunology Research Center, Institute of Research for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran.
- 2 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
- PMID: 31819702
- PMCID: PMC6885567
- DOI: 10.2147/BLCTT.S230383
Abstract
Introduction: Human Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) which encodes survivin exhibits multiple biological activities, such as cell proliferation and apoptosis. Survivin is overexpressed in numerous malignant diseases including acute myeloid leukemia (AML). Recent studies have shown that the CRISPR/Cas9 nuclease-mediated gene-editing systems are suitable approach’s for editing or knocking out various genes including oncogenes.
Methods and materials: We used CRISPR-Cas9 to knockout the BIRC5 in the human leukemic cell line, HL60, and KG1, and these cell lines were transfected with either the Cas9- and three sgRNAs expressing plasmids or negative control (scramble) using Lipofectamine 3000. The efficacy of the transfection was determined by quantitative reverse transcription-polymerase chain (RT-qPCR) and surveyor mutation assays. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively.
Results: We have successfully knocked out the BIRC5 gene in these leukemic cells and observed that the BIRC5-knocked out cells by CRISPR/Cas9 showed a significant decrease (30 folds) of survivin at mRNA levels. Moreover, cell death and apoptosis were significantly induced in BIRC5-CRISPR/Cas9-transfected cells compared to the scramble vector.
Conclusion: We demonstrated for the first time that targeting BIRC5 by CRISPR/Cas9 technology is a suitable approach for the induction of apoptosis in leukemic cells. However, further studies targeting this gene in primary leukemic cells are required.
Keywords: AML; BIRC5; CRISPR/Cas9 nuclease; HL60 cell; KG1 cells; survivin.
© 2019 Narimani et al.
Conflict of interest statement
This work was supported by grants from Kurdistan University of Medical Sciences to AJ to purchasing reagents for the thesis of MN as a PhD candidate in Molecular Medicine. The authors report no other conflicts of interest in this work.
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