LIU Kai1, SUN Xin-di1, ZHANG Wei-wei1.2, YANG Qing-zhu1.2, HUANG Xin1.2, SHAO Shu-li1.2
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
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.
PDF 
Down-regulation of MDR1 gene expression by CRISPRi to enhance the sensitivity of lung adenocarcinoma A549/DDP cells to cisplatin.pdf