利用CRISPRi下调 MDR1基因表达增强肺腺癌 A549/DDP细胞对顺铂敏感性*

doi:10.12047/j.cjap.6342.2022.109

中国应用生理学杂志 ›› 2022, Vol. 38 ›› Issue (5): 590-594.doi: 10.12047/j.cjap.6342.2022.109

• 研究论文 • 上一篇

利用CRISPRi下调 MDR1基因表达增强肺腺癌 A549/DDP细胞对顺铂敏感性^*

刘凯¹, 孙新迪¹, 张伟伟^1.2, 杨清竹^1.2, 黄鑫^1.2, 邵淑丽^1.2△

1.齐齐哈尔大学生命科学与农林学院;
2.抗性基因工程与寒地生物多样性保护黑龙江省重点实验室, 黑龙江齐齐哈尔 161006

收稿日期:2022-07-22 修回日期:2022-09-28 出版日期:2022-09-28 发布日期:2023-04-23
通讯作者: ^△Tel: 0452-2738219; E-mail: shshl32@163.com
基金资助:
^*国家自然科学基金项目(31670843);黑龙江省教育厅基本业务专项重点项目(135109104);黑龙江省教育厅基本业务专项(粮头食尾)(LTSW201737);黑龙江省省属高等学校基本科研业务费科研项目(植物性食品加工技术特色学科专项,YSTSXK201809);齐齐哈尔大学2021年研究生创新科研项目(YJSCX2021029)

Down-regulation of MDR1 gene expression by CRISPRi to enhance the sensitivity of lung adenocarcinoma A549/DDP cells to cisplatin

LIU Kai¹, SUN Xin-di¹, ZHANG Wei-wei^1.2, YANG Qing-zhu^1.2, HUANG Xin^1.2, SHAO Shu-li^1.2△

1. College of Life Science and Agroforestry, Qiqihar University;
2. Heilongjiang Key Laboratory of Resistance Genetic Engineering and Biodiversity Protection in Cold Regions, Qiqihar 161006, China

Received:2022-07-22 Revised:2022-09-28 Online:2022-09-28 Published:2023-04-23

摘要/Abstract

摘要： 目的: 探讨利用CRISPRi下调 MDR1基因表达增强肺腺癌 A549/DDP细胞对顺铂敏感性的作用。方法: 利用生物信息学技术预测MDR1基因启动子上潜在的CRISPRi干扰位点,设计干扰片段并构建重组载体,采用qRT-PCR、Western blot方法检测各组细胞中MDR1基因mRNA以及蛋白表达水平,筛选干扰效率高的重组载体进行后续实验。将人肺癌A549/DDP细胞分为3组,分别为A549/DDP、Scrambed、sgRNA-MDR1-1,每组设置3个复孔。将各载体转染细胞48 h后,流式细胞术检测各组细胞外排情况,MTT法检测各组细胞的IC₅₀值,激光共聚焦显微镜下观察各组细胞经顺铂处理后的细胞形态。结果: 经测序比对,成功构建两种干扰MDR1基因转录的CRISPRi重组载体。转染A549/DDP细胞后,各转染组细胞MDR1基因mRNA以及蛋白水平均显著降低(P＜ 0.01),其中sgRNA-MDR1-1的干扰效率最高,mRNA和蛋白水平干扰效率分别达60%和51%。与Scrambed组相比,转染sgRNA-MDR1-1组细胞的细胞外排能力降低(P＜0.01),细胞对顺铂的IC₅₀值显著降低(P＜0.01),细胞内染色质聚集边缘化。结论: 利用CRISPRi技术干扰MDR1基因表达能增强肺腺癌A549/DDP细胞对顺铂的敏感性。

关键词: CRISPRi, MDR1基因, 药物敏感性, 肺腺癌 A549/DDP细胞, 细胞培养

Abstract: Objective: To investigate the effects of down-regulating MDR1 gene expression by CRISPRi on enhancing the sensitivity of lung adenocarcinoma A549/DDP cells to cisplatin. Methods: The potential CRISPRi interference sites on the MDR1 gene promoter were predicted by bioinformatics software, and the interference fragments were designed and constructed. The mRNA and protein expression levels of MDR1 gene in each group of cells were detected by qRT-PCR and Western blot methods, and the recombinant vectors with high interference efficiency were screened. Human lung cancer A549/DDP cells were divided into three groups: A549/DDP, Scrambed and sgRNA-MDR1-1, with three multiple holes in each group. After each vector was transfected into the cells for 48 h, the efflux of cells in each group was detected by flow cytometry, the IC₅₀ value of cells in each group was detected by MTT method, and the cell morphology of cells treated with cisplatin was observed under laser confocal microscope. Results: After sequencing and comparison, two kinds of CRISPRi recombinant vectors interfering with MDR1 gene transcription were constructed successfully. After transfection of A549/DDP cells, the mRNA and protein levels of MDR1 gene in all transfection groups were decreased significantly (P＜ 0.01). Among them, the interference efficiency of sgRNA-MDR1-1 was the highest, and the interference efficiency of mRNA and protein was 60% and 51%, respectively. After transfection of sgRNA-MDR1-1 vector, compared with the control group, the efflux ability of cells was decreased (P＜0.01), the IC₅₀ value of cells to cisplatin was decreased significantly (P＜0.01), and the intracellular chromatin gathered and marginalized, and apoptotic bodies appeared. Conclusion: CRISPRi interference with MDR1 gene in drug-resistant A549/DDP cells can significantly enhance the sensitivity to cisplatin.

Key words: CRISPRi, MDR1 gene, drug sensitivity, lung adenocarcinoma A549/DDP cell, cell culture

中图分类号:

Q291

刘凯, 孙新迪, 张伟伟, 杨清竹, 黄鑫, 邵淑丽. 利用CRISPRi下调 MDR1基因表达增强肺腺癌 A549/DDP细胞对顺铂敏感性^*[J]. 中国应用生理学杂志, 2022, 38(5): 590-594.

LIU Kai, SUN Xin-di, ZHANG Wei-wei, YANG Qing-zhu, HUANG Xin, SHAO Shu-li. Down-regulation of MDR1 gene expression by CRISPRi to enhance the sensitivity of lung adenocarcinoma A549/DDP cells to cisplatin[J]. CJAP, 2022, 38(5): 590-594.

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利用CRISPRi下调 MDR1基因表达增强肺腺癌 A549/DDP细胞对顺铂敏感性^*

Down-regulation of MDR1 gene expression by CRISPRi to enhance the sensitivity of lung adenocarcinoma A549/DDP cells to cisplatin

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