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Evaluation of CRISPR/Cas9 System Effects on Knocking Out NEAT1 Gene in AGS Gastric Cancer Cell Line with Therapeutic Perspective

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Abstract

Aim

Gastric cancer (GC) is one of the most common malignant tumors globally, with an increasing incidence rate. Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long non-coding RNA (lncRNAs) responsible for regulating cell cycle progression, apoptosis, cell growth, proliferation, and migration in various cells. The present survey was performed to assess the effects of NEAT1 gene knocking out by CRISPR/Cas9 system in human gastric cancer cells.

Methods

The CRISPR/Cas9 genome editing technique was used to knockout NEAT1 in AGS cells as a gastric cancer model. After the design and construction of the vector, transfection was performed. The expression levels of mRNA, the survival of cells, apoptosis, and cell migration were evaluated by real-time quantitative polymerase chain reaction, flow cytometry, and scratch wound.

Results

Degradation of NEAT1 by CRISPR/cas9 significantly suppressed the gene’s expression rate, arrested cell cycle in the G0/G1 phase, and a significant reduction in cell number in the S phase (P < 0.05). Degradation of NEAT1 by CRISPR/cas9 also restrained the ability to migrate in transfected cells compared to the control group (P < 0.01). Knockout of NEAT1 via impact on miR-34a gene expression induced apoptosis of AGS cells (P < 0.05) with increasing in the FAS level and total apoptosis (P < 0.001).

Conclusions

Findings suggest that NEAT1 plays a vital role in cellular mechanisms of GC’s occurrence and can serve as a new treatment target in GC.

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Acknowledgements

The results presented in this paper were part of the Ph.D student thesis and were financially supported by personal student cost.

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Authors and Affiliations

  1. Department of Genetics, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran

    Nastaran Haghighi

  2. Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran

    Abbas Doosti

  3. Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences (IUMS), Tehran, Iran

    Jafar Kiani

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  1. Nastaran Haghighi
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  2. Abbas Doosti
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  3. Jafar Kiani
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Correspondence to Abbas Doosti.

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Haghighi, N., Doosti, A. & Kiani, J. Evaluation of CRISPR/Cas9 System Effects on Knocking Out NEAT1 Gene in AGS Gastric Cancer Cell Line with Therapeutic Perspective. J Gastrointest Canc 53, 623–631 (2022). https://doi.org/10.1007/s12029-021-00669-z

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  • DOI: https://doi.org/10.1007/s12029-021-00669-z

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