Stable knockout of ACSS3 in lung cancer cell line using CRISPR/Cas 9 technology

doi:10.16352/j.issn.1001-6325.2025.08.1016

Abstract

Abstract: Objective To explore the effect of acyl-CoA synthetase short-chain family member 3(ACSS3) gene on the proliferation of human large cell lung cancer cells(NCI-H460) using CRISPR/Cas 9 gene editing technology. Methods The expression of ACSS3 was detected by Western blot. ACSS3-targeting sgRNAs were designed, and a CRISPR/Cas 9 knockout vector was constructed and transfected into NCI-H460 cells. The transfected cells were selected with puromycin based on vector-carried resistance. ACSS3-knockout monoclonal cell strains were established by limited dilution method and then expanded in culture. Knockout efficiency was confirmed by Western blot. Cell proliferation was assessed using MTT and colony formation assays. Results The expression of ACSS3 was significantly elevated in NCI-H460 cells as compared with human normal lung epithelial cells BEAS-2B(P＜0.05). No ACSS3 protein was detected in the knockout monoclonal strain, indicating successful generation of ACSS3-knockout NCI-H460 cells. Compared with the control cells transfected with empty vector, the proliferation and colony formation ability were inhibited in NCI-H460 cells with ACSS3 knockout(P＜0.05). Conclusions The ACSS3-knockout NCI-H460 cell strain was successfully established, which provides a foundation for further study on the role of ACSS3 in lung cancer.

Key words: lung cancer, CRISPR/Cas 9, ACSS3

CLC Number:

HUANG Qianqian, JIA Yufang, YU Huajun,CHEN Rongrong,CHEN Lili, WU Jun, ZHANG Haitao. Stable knockout of ACSS3 in lung cancer cell line using CRISPR/Cas 9 technology[J]. Basic & Clinical Medicine, 2025, 45(8): 1016-1021.

References

[1] Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024[J]. CA Cancer J Clin, 2024, 74: 12-49.
[2] Li Y, Yan B, He S. Advances and challenges in the treatment of lung cancer[J]. Biomed Pharmacother, 2023, 169: 115891. doi:10.1016/j.biopha.2023.115891.
[3] Zhang Q, Shi Y, Liu S, et al. EZH2/G9a interact to mediate drug resistance in non-small-cell lung cancer by regulating the SMAD4/ERK/c-Myc signaling axis[J]. Cell Rep, 2024, 43: 113714. doi:10.1016/j.celrep.2024.113714.
[4] Miller KD, O'connor S, Pniewski KA, et al. Acetate acts as a metabolic immunomodulator by bolstering T-cell effector function and potentiating antitumor immunity in breast cancer[J]. Nat Cancer, 2023, 4: 1491-1507.
[5] Yoshimura Y, Araki A, Maruta H, et al. Molecular cloning of rat acss3 and characterization of mammalian propionyl-CoA synthetase in the liver mitochondrial matrix[J]. J Biochem, 2017, 161: 279-289.
[6] Jia Z, Chen X, Chen J, et al. ACSS3 in brown fat drives propionate catabolism and its deficiency leads to autophagy and systemic metabolic dysfunction[J]. Clin Transl Med, 2022, 12: e665. doi:10.1002/ctm2.665.doi: 10.3389/fphys.2017.00519.
[7] Chang WC, Cheng WC, Cheng BH, et al. Mitochondrial acetyl-CoA synthetase 3 is biosignature of gastric cancer progression[J]. Cancer Med, 2018, 7: 1240-1252.
[8] Zhou L, Song Z, Hu J, et al. ACSS3 represses prostate cancer progression through downregulating lipid droplet-associated protein PLIN3[J]. Theranostics, 2021, 11: 841-860.
[9] Zhang J, Duan H, Feng Z, et al. Acetyl-CoA synthetase 3 promotes bladder cancer cell growth under metabolic stress[J]. Oncogenesis, 2020, 9: 46. doi:10.1038/s41389-020-0230-3.
[10] Wang L, Yuan H, Li W, et al. ACSS3 regulates the metabolic homeostasis of epithelial cells and alleviates pulmonary fibrosis[J]. Biochim Biophys Acta Mol Basis Dis, 2024, 1870: 166960. doi:10.1016/j.bbadis.2023.166960.
[11] Rodríguez-Rodríguez DR, Ramírez-Solís R, Garza-Elizondo MA, et al. Genome editing:a perspective on the applica-tion of CRISPR/Cas9 to study human diseases(Review)[J]. Int J Mol Med, 2019, 43: 1559-1574.
[12] Klermund J, Rhiel M, Kocher T, et al. On- and off-target effects of paired CRISPR-Cas nickase in primary human cells[J]. Mol Ther, 2024, 32: 1298-1310.
[13] Lorenzo D, Esquerda M, Palau F, et al. Ethics and genomic editing using the Crispr-Cas9 technique: challenges and conflicts[J]. NanoEthics, 2022, 16: 313-321.
[14] Manghwar H, Li B, Ding X, et al. CRISPR/Cas systems in genome editing: methodologies and tools for sgRNA design, off-target evaluation, and strategies to mitigate off-target effects[J]. Adv Sci(Weinh), 2020, 7: 1902312.doi:10.1002/advs.201902312.

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