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

10.30498/ijb.2022.310632.3180

Abstract

Background: lncRNAs play an important role in cellular mechanisms, including transcription, translation, and apoptosis. NEAT1 is one of the most important types of lncRNAs in humans that are able to bind to active genes. increased its expression have been shown in a variety of cancers, including kidney cancer. Kidney cancer accounts for approximately 3% of all cancers in the world and occurs almost twice as often in men as in women.

Objectives: This study has been performed to knock out the NEAT1 gene using the CRISPR/Cas9 technique in an ACHN cell line and to evaluate its effects on cancer progression and apoptosis.

Material and Methods: Two specific 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 used to evaluate the survival of cells, cell proliferation and cell migration, respectively.

Results: The results have showed successful degradation of the NEAT1 gene in the cells of the treatment group. Expression of the P53, BAK, BAX and FAS genes in the cells of the treatment group (NEAT1 knockout) showed a significant increase 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: Degeneration of the NEAT1 gene in the ACHN cell line using the new CRISPR/Cas9 technique showed satisfactory effects in reducing cell survival and proliferation and increasing apoptosis. Therefore, it seems that the degradation of the NEAT1 gene has an effective role in controlling the growth of kidney cancer cells.

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