Evaluation of Apoptosis, Cell Proliferation and Cell Cycle Progression by Inactivation of the NEAT1 Long Noncoding RNA in a Renal Carcinoma Cell Line Using CRISPR/Cas9

Document Type : Research Paper

Authors

1 Department of Biology, Faculty of Basic Sciences, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.

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

3 Department of Biology, Faculty of Basic Sciences, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran. Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences (IUMS), Tehran, Iran.

10.30498/ijb.2022.310632.3180

Abstract

Background: Long noncoding RNAs (lncRNAs) play an important role in cellular mechanisms including transcription, translation, and apoptosis. NEAT1 is one of the essential types of lncRNAs in humans that can bind to active genes and modify their transcription. NEAT1 upregulation in various forms of cancer such as kidney cancer has been reported. Kidney cancer accounts for approximately 3% of all cancers worldwide and occurs almost twice as often in men as in women.
Objectives: This study has been performed to knockout the NEAT1 gene using the CRISPR/Cas9 technique in the Renal Cell Carcinoma ACHN cell line and to evaluate its effects on cancer progression and apoptosis.
Material and Methods: Two specific (single guide RNA (sgRNA) sequences for the NEAT1 gene were designed by CHOPCHOP software. These sequences were then cloned into plasmid pSpcas9, and recombinant vectors PX459-sgRNA1 and PX459-sgRNA2 were generated. ACHN cells were transfected using recombinant vectors carrying sgRNA1 and sgRNA2. The expression level of apoptosis-related genes was assessed by real-time PCR. Annexin, MTT and cell scratch tests were performed to evaluate the survival, proliferation, and migration of the knocked out cells, respectively.
Results: The results have shown successful knockout of the NEAT1 gene in the cells of the treatment group. Expressions of P53, BAK, BAX and FAS genes in the cells of the treatment group (NEAT1 knockout) showed significant increases in expression compared to the cells of the control group (P <0.01). Additionally, decreased expression of BCL2 and survivin genes was observed in knockout cells compared to the control group (p <0.05). In addition, in the cells of the treatment group compared to control cells, a significant decrease in cell viability, ability to migrate and cell growth and proliferation was observed.
Conclusion: Inactivation of the NEAT1 gene in ACHN cell line using CRISPR/Cas9 technology elevated apoptosis and reduced cell survival and proliferation which makes it a novel target for kidney cancer therapeutics.

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