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.
Highlights
Hematological malignancies are a complex and divergent group of diseases caused by abnormality in hematopoietic cells and categorized into three types of leukemia, lymphoma, and myeloma[1]
Cell culture This study included two different types of Acute myelocytic leukemia (AML) cell lines: human KG-1 cancer cell line, established from bone marrow cells of a patient with erythroleukemia evolving to acute myelogenous leukemia[28], and HL60 cells, which were derived from a 36-year-old woman[29]
baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) gene is located on the telomeric position of chromosome 17q25.3 in forward strand, and 11 transcripts has been detected in this gene; six of them are transcribed to protein and three of which, BIRC5-2B, BIRC5-WT, and BIRC5-deltaEx3, are the most important
Summary
Hematological malignancies are a complex and divergent group of diseases caused by abnormality in hematopoietic cells and categorized into three types of leukemia, lymphoma, and myeloma[1]. Acute myelocytic leukemia (AML) is a clonal malignancy resulting from the aggregation of immature myeloid precursors called blast, due to the occurrence of more than one driver genetic mutations[4,5]. 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. 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.
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