CRISPRi技术沉默MRP1基因表达增强A549/DDP细胞对TSN的敏感性  

CRISPRi technology silences MRP1 gene expression and enhances the sensitivity of A549/DDP cells to TSN

作　　者：孟令雪 郭旭 孙新迪[1] 沈洋 张伟伟[1,2] 杨清竹 邵淑丽[1,2] MENG Lingxue;GUO Xu;SUN Xindi;SHEN Yang;ZHANG Weiwei;YANG Qingzhu;SHAO Shuli(School of Life Sciences,Agriculture and Forestry,Qiqihar 161006,China;School of Life Sciences,Heilongjiang Provincial Key Laboratory of Resistance Gene Engineering and Protection of Biodiversity in Cold Areas,Qiqihar University,Qiqihar 161006,China)

机构地区：[1]齐齐哈尔大学生命科学与农林学院,黑龙江齐齐哈尔161006 [2]齐齐哈尔大学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

出　　处：《高师理科学刊》2022年第7期66-70,85,共6页Journal of Science of Teachers＇College and University

基　　金：齐齐哈尔大学黑龙江省教育厅基本业务专项重点项目(135109104);黑龙江省高教强省优势特色学科——玉米“粮头食尾”重点项目(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);2020年齐齐哈尔大学研究生创新科研项目(YJSCX2020046);国家自然科学基金项目(31670843)。

摘　　要：利用CRISPRi技术构建靶向MRP1的干扰载体,采用生物信息学分析MRP1的启动子序列,设计并合成3对sgRNA干扰片段,构建3种靶向MRP1的干扰表达载体,分别转染A549和A549/DDP细胞后,通过qRT-PCR和Western blot方法检测MRP1 mRNA和蛋白的表达情况,MTT法检测细胞对川楝素的敏感性,激光共聚焦显微镜下观察细胞的形态学变化.结果表明,基因测序证实成功构建了3种干扰载体,分别转染干扰载体至A549/DDP细胞48 h后MRP1 RNA和蛋白表达水平均显著降低(P<0.01),其中sgRNA-MRP1-2干扰效果最好;将sgRNA-MRP1-2转染并使用60 nmol/L川楝素处理后,细胞对川楝素的敏感性显著增加,IC50值显著降低(P<0.01),细胞形态由梭形变为椭圆形,染色质高度凝聚、边缘化.The MRP1-targeting interference vector was constructed by CRISPRi technology,the promoter sequence of MRP1 was analyzed by bioinformatics,3 pairs of sgRNA interference fragments were designed and synthesized,and 3 MRP1-targeting interference expression vectors were constructed and transfected with A549 and A549/DDP respectively.After cells,the expression of MRP1 mRNA and protein was detected by qRT-PCR and Western blot methods,the sensitivity of cells to toosendanin was detected by MTT method,and the morphological changes of cells were observed under a laser confocal microscope.The results showed that three kinds of interference vectors were successfully constructed by gene sequencing,and the expression levels of MRP1 RNA and protein were significantly decreased(P<0.01)after transfecting the interference vectors into A549/DDP cells for 48 hours respectively(P<0.01),among which sgRNA-MRP1-2 had the most interference effect.After sgRNA-MRP1-2 was transfected and treated with 60 nmol/L toosendanin,the sensitivity of cells to toosendanin was significantly increased,the IC50 value was significantly decreased(P<0.01),and the cell shape changed from spindle to oval,chromatin is highly condensed and marginalized.

关 键 词：CRISPRi MRP1 细胞培养 人肺癌A549/DDP细胞 

分 类 号：Q81[生物学—生物工程]