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

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IC 50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IC 50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。

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 IC 50 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 IC 50 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.