BIRC5 Gene Disruption via CRISPR/Cas9n Platform Suppress Acute Myelocytic Leukemia Progression

Narimani, Manizheh; Sharifi, Mohammadreza; Hakhamaneshi, Mohammad Saeed; Roshani, Daem; Kazemi, Mohammad; Hejazi, Seyed Hossein; Jalili, Ali

doi:10.29252/ibj.23.6.369

BIRC5 Gene Disruption via CRISPR/Cas9n Platform Suppress Acute Myelocytic Leukemia Progression

Authors

Manizheh Narimani ¹

Mohammadreza Sharifi ²

Mohammad Saeed Hakhamaneshi ¹

Daem Roshani ¹

Mohammad Kazemi ²

Seyed Hossein Hejazi ³

Ali Jalili ¹

¹ Cancer and Immunology Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran

² Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

³ Skin Diseases and Leishmaniasis Research Center, Department of Parasitology & Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

10.29252/ibj.23.6.369

Abstract

Background: Acute myelocytic leukemia (AML) is a clonal malignancy resulting from the accumulation of genetic abnormalities in the cells. Human baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), encodes survivin, is one of only a handful of genes that is differentially over-expressed in numerous malignant diseases including AML. Methods: The BIRC5 was silenced permanently in two AML cell lines, HL‑60 and KG-1, via the CRISPR/Cas9n system. After transfection of CRISPR constructs, genomic DNA was extracted and amplified to assess mutation detection. To evaluate BIRC5 gene expression, quantitative real-time PCR was performed. Also, MTT cell viability and Annexin‑V/propidium iodide flowcytometric staining were performed, and the data were analyzed using the Kolmogorov-Smirnov, Levene's, and ANOVA tests. Results: The results indicated that Cas9n and its sgRNAs successfully triggered site-specific cleavage and mutation in the BIRC5 gene locus. Moreover, suppression of BIRC5 resulted in the reduction of cell viability, and induction of apoptosis and necrosis in HL60 and KG1 suggested that the permanent suppression of BIRC5 remarkably dropped the gene expression and cells viability. Conclusion: This study reinforces the idea that BIRC5 disruption via Cas9n:sgRNAs has favorable effects

on the AML clinical outcome. It thereby can be a promising candidate in a variety of leukemia treatments.

Keywords

Acute myelocytic leukemia (AML)

CRISPR

Gene editing

Survivin