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Knockout Of BIRC5 Gene By CRISPR/Cas9 Induces Apoptosis And Inhibits Cell Proliferation In Leukemic Cell Lines, HL60 And KG1.

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.

SUBMITTER: Narimani M

PROVIDER: S-EPMC6885567 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Knockout Of <i>BIRC5</i> Gene By CRISPR/Cas9 Induces Apoptosis And Inhibits Cell Proliferation In Leukemic Cell Lines, HL60 And KG1.

Narimani Manizheh M Sharifi Mohammadreza M Jalili Ali A

Blood and lymphatic cancer : targets and therapy 20191127

<h4>Introduction</h4>Human Baculoviral inhibitor of apoptosis repeat-containing 5 (<i>BIRC5</i>) 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.<h4>Methods and materials</h ...[more]

PMID: 31819702

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Project description:BackgroundThe growing detection of long noncoding RNAs (lncRNAs) required the application of functional approaches in order to provide absolutely precise, conducive, and reliable processed information along with effective consequences. We utilized genetic knockout (KO) techniques to ablate the Long Intergenic Noncoding RNA 00,511 gene in several humans who suffered from breast cancer cells and at the end we analyzed and examined the results.ResultsThe predictive relevance of LINC00511 expression pattern was measured by using a pooled hazard ratio (HR) with a 95% confidence interval (CI). The link among LINC00511 expression profiles and cancer metastasis was measured by using a pooled odds ratio (OR) with a 95% confidence interval. This meta- analysis was composed of fifteen studies which contained a total of 1040 tumor patients. We used three distinct CRISPR/Cas9-mediated knockdown techniques to prevent the LINC00511 lncRNA from being transcribed. RT-PCR was used to measure lncRNA and RNA expression. We used CCK-8, colony formation tests, and the invasion transwell test to measure cell proliferation and invasion. The stemness was measured by using a sphere-formation test. To validate molecular attachment, luciferase reporter assays were performed. The functional impacts of LINC00511 gene deletion in knockdown breast cancer cell lines were confirmed by using RT-qPCR, MTT, and a colony formation test. This meta-analysis was composed of 15 trials which contained a total of 1040 malignant tumors. Greater LINC00511 expression was ascribed to a lower overall survival (HR = 1.93, 95% CI 1.49-2.49, < P 0.001) and to an increased proportion of lymph node metastasis (OR = 3.07, 95% CI 2.23-4.23, P < 0.001) in the meta-analysis. It was found that the role of LINC00511 was overexpressed in breast cancer samples, and this overexpression was ascribed to a poor prognosis. The gain and loss-of-function tests demonstrated findings such as LINC00511 increased breast cancer cell proliferation, sphere-forming ability, and tumor growth. Additionally, the transcription factor E2F1 binds to the Nanog gene's promoter site to induce transcription. P57, P21, Prkca, MDM4, Map2k6, and FADD gene expression in the treatment group (LINC00511 deletion) was significantly higher than in the control group (P < 0.01). In addition, knockout cells had lower expression of BCL2 and surviving genes than control cells P < 0.001). In each of the two target alleles, the du-HITI approach introduced a reporter and a transcription termination signal. This strategy's donor vector preparation was significantly easier than "CRISPR HDR," and cell selection was likewise much easier than "CRISPR excision." Furthermore, when this approach was used in the initial transfection attempt, single-cell knockouts for both alleles were generated.ConclusionsThe methods employed and described in this work could be extended to the production of LINC00511 knockout cell lines and, in theory, to the deletion of other lncRNAs to study their function.

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Knockout Of BIRC5 Gene By CRISPR/Cas9 Induces Apoptosis And Inhibits Cell Proliferation In Leukemic Cell Lines, HL60 And KG1.

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Knockout Of <i>BIRC5</i> Gene By CRISPR/Cas9 Induces Apoptosis And Inhibits Cell Proliferation In Leukemic Cell Lines, HL60 And KG1.

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