用 CRISPR/Cas 9技术构建ACSS3基因稳定敲除的肺癌细胞株

doi:10.16352/j.issn.1001-6325.2025.08.1016

基础医学与临床 ›› 2025, Vol. 45 ›› Issue (8): 1016-1021.doi: 10.16352/j.issn.1001-6325.2025.08.1016

用 CRISPR/Cas 9技术构建ACSS3基因稳定敲除的肺癌细胞株

黄倩倩^1,2, 贾玉芳^2,3, 余华军², 陈融融², 陈丽丽^2,4, 伍俊⁴, 张海涛^2*

广东医科大学 1.医学技术学院;2.生物化学与分子生物学研究所&多肽和蛋白研究与应用重点实验室,广东湛江 524023;
3.西安大兴医院检验科,陕西西安 710016;
4.广东医科大学附属医院呼吸内科,广东湛江 524023

收稿日期:2024-07-05 修回日期:2024-11-22 出版日期:2025-08-05 发布日期:2025-07-11
通讯作者: ^*taohaizhang33@163.com
基金资助:
广东省基础与应用基础研究基金自然科学基金项目(2024A1515013157);湛江市科技发展专项资金竞争性分配项目(2022A702-1)

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

HUANG Qianqian^1,2, JIA Yufang^2,3, YU Huajun²,CHEN Rongrong²,CHEN Lili^2,4, WU Jun⁴, ZHANG Haitao^2*

1. School of Medical Technology; 2. Institute of Biochemistry and Molecular Biology & Key Laboratory of Peptide and Protein Research and Application, Guangdong Medical University, Zhanjiang 524023;
3. Department of Clinical Laboratory, Xi'an Daxing Hospital, Xi'an 710016;
4. Department of Respiratory Medicine, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524023, China

Received:2024-07-05 Revised:2024-11-22 Online:2025-08-05 Published:2025-07-11
Contact: ^*taohaizhang33@163.com

摘要/Abstract

摘要： 目的利用CRISPR/Cas 9基因编辑技术研究酰基辅酶A合成酶短链家族成员3(ACSS3)基因对人大细胞肺癌细胞(NCI-H460)增殖的影响。方法用Western blot检测ACSS3在肺癌细胞中的表达。设计sgRNA,构建CRISPR/Cas 9靶向敲除载体并转染NCI-H460细胞。用嘌呤霉素筛选出成功转染的细胞,用有限稀释法筛选出稳定敲除ACSS3的单克隆细胞株并扩大培养。Western blot 验证敲除细胞株ACSS3的表达。MTT法和集落形成实验检测敲除ACSS3基因对NCI-H460细胞增殖的影响。结果与人正常肺上皮细胞BEAS-2B细胞相比,NCI-H460细胞中ACSS3的表达量显著升高(P＜0.05)。Western blot检测敲除细胞株中ACSS3表达呈阴性。与空载体转染的对照组细胞相比,敲除ACSS3后,NCI-H460细胞的增殖活力和集落形成能力受到抑制(P＜0.05)。结论成功获得稳定敲除ACSS3基因的NCI-H460细胞株,为后续研究ACSS3在肺癌中的作用机制奠定基础。

关键词: 肺癌, CRISPR/Cas 9, ACSS3

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

中图分类号:

黄倩倩, 贾玉芳, 余华军, 陈融融, 陈丽丽, 伍俊, 张海涛. 用 CRISPR/Cas 9技术构建ACSS3基因稳定敲除的肺癌细胞株[J]. 基础医学与临床, 2025, 45(8): 1016-1021.

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.

参考文献

[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.

[1]	陈磊, 任伟豪, 王立德. 敲降线粒体内膜转运酶8A提升肺癌细胞系PC-9对吉非替尼治疗的敏感性[J]. 基础医学与临床, 2025, 45(8): 1073-1077.
[2]	杨瑞, 杜斯奋, 蒋乐辉, 付恬, 任鹏举, 蒋澄宇, 张艳丽. 基于网络药理学的抗肺癌茶叶小分子筛选及验证[J]. 基础医学与临床, 2025, 45(7): 939-946.
[3]	楼海均, 童卓云, 张振宇, 阿合叶尔克·马合沙提, 孟·孟根, 乌都木丽. 敲低DRAM2抑制肺癌细胞系A549的增殖和迁移[J]. 基础医学与临床, 2025, 45(2): 197-202.
[4]	郑红, 陈晓霞, 韩李周, 陈苗, 皇甫娟. 降低lncRNA-RMRP表达抑制人肺癌细胞系A549的增殖和侵袭[J]. 基础医学与临床, 2024, 44(7): 974-978.
[5]	操辰新, 唐辉, 耿瑞璇, 郭伏平, 白春梅, 王颖轶, 李太生. 循环CD4⁺CD45RA⁺CD62L⁺ T细胞与接受EGFR-TKI治疗的转移性非小细胞肺癌预后相关[J]. 基础医学与临床, 2024, 44(5): 658-664.
[6]	梁香存, 魏小雨, 梁健, 王庆, 耿广. 安罗替尼通过调控miR-16-5p/PD-1轴抑制人非小细胞肺癌细胞系增殖并促进凋亡[J]. 基础医学与临床, 2024, 44(4): 503-512.
[7]	张婧, 杨自更, 蔡文琴, 曹维维, 韦红梅, 薛茜茜, 吴宾. 抑制抗增殖蛋白2增强非小细胞肺癌细胞系A549对厄洛替尼敏感性[J]. 基础医学与临床, 2024, 44(3): 325-332.
[8]	李娟, 李晓峰, 徐生志, 唐凯. 华蟾素胶囊联合多西紫杉醇+顺铂化疗用于非小细胞肺癌患者的疗效[J]. 基础医学与临床, 2024, 44(2): 247-251.
[9]	丁飞, 王洒, 黄常新. 非小细胞肺癌患者血清CA211水平和NK细胞数与预后相关[J]. 基础医学与临床, 2024, 44(2): 180-184.
[10]	贾坤, 周妍妍. MYC通过下调AKR1C1表达抑制肺癌细胞系迁移[J]. 基础医学与临床, 2024, 44(12): 1621-1627.
[11]	张伟, 张振, 杨连任, 梁伟. 肺癌7项抗体水平与NSCLC患者化疗效果及预后的关系[J]. 基础医学与临床, 2024, 44(10): 1383-1387.
[12]	王春燕, 王萍, 宋龙飞, 刘永全, 满君. LncRNA FEZF1-AS1通过调控EZH2对肺间质细胞增殖、迁移及侵袭的作用[J]. 基础医学与临床, 2024, 44(1): 43-50.
[13]	颜晨红, 金儿. 外泌体PD-L1在非小细胞肺癌诊断和治疗上的研究进展[J]. 基础医学与临床, 2023, 43(9): 1457-1461.
[14]	张小婉, 康霞, 姚小英, 谢芳, 李莹. 沉默SF3B1促进人肺癌细胞系A549凋亡并抑制其增殖及侵袭[J]. 基础医学与临床, 2023, 43(8): 1265-1270.
[15]	李珍, 段昭君, 罗云萍. 神经节苷脂GD2可作为肺癌干细胞一个潜在的候选标志物[J]. 基础医学与临床, 2023, 43(5): 777-784.

用 CRISPR/Cas 9技术构建ACSS3基因稳定敲除的肺癌细胞株

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价