可诱导型敲低PICH的MDA-MB-231细胞系的构建及鉴定  

Establishment and Identification of MDA-MB-231 Cell Line with Stable Expression of Inducible PICH shRNA

作　　者：李万金 黄燕 吕璐冶 韩秋影[1] 周涛[1] 陈亮[1] LI Wan-Jin;HUANG Yan;LYU Lu-Ye;HAN Qiu-Ying;ZHOU Tao;CHEN Liang(National Center of Biomedical Analysis,Beijing 100850,China)

机构地区：[1]国家生物医学分析中心,北京100850

出　　处：《生物技术通讯》2019年第2期153-157,共5页Letters in Biotechnology

基　　金：国家自然科学基金(81872153)

摘　　要：目的:构建稳定表达可诱导型polo样激酶1相关检查点解旋酶(PICH)短发夹RNA(shRNA)的三阴乳腺癌细胞系MDA-MB-231,并通过检测加入诱导剂后细胞的增殖评价敲低效果。方法:设计并合成2条特异靶向PICH的shRNA序列,用分子克隆技术构建pLKO.1-tet-on-shPICH-Puromycin质粒,经菌落PCR及基因测序验证;慢病毒包装并感染细胞,通过抗性筛选获得嘌呤霉素抗性的细胞;经强力霉素(DOX)诱导表达后,Western印迹检测PICH的敲低效果,并通过MTT法对细胞增殖进行检测。结果:菌落PCR凝胶电泳及DNA测序结果显示pLKO.1-tet-on-shPICHPuromycin质粒构建成功;Western印迹结果表明,在DOX诱导下,MDA-MB-231细胞中的PICH蛋白质表达下降,表明可诱导型PICH稳定敲低细胞系构建成功;进一步实验结果显示,MDA-MB-231细胞的增殖随PICH的表达下调而受到显著抑制。结论:构建了可诱导稳定敲低PICH的三阴乳腺癌细胞系MDA-MB-231,为进一步研究PICH在三阴乳腺癌中的作用以及具体的分子机制奠定了实验基础。Objective:To generate triple-negative breast cancer MDA-MB-231 cell line stably expressing inducible short hairpin RNA(shRNA)targeting polo-like kinase 1-interacting checkpoint helicase(PICH).Methods:Two oligonucleotides targeting PICH were synthesized and inserted separately into pLKO.1-tet-on-Puromycin vectors.The pLKO.1-tet-on-shPICH-Puromycin plasmids were validated by PCR and sequencing.Lentivirus expressing control or PICH-targeting shRNA was used to infect MDA-MB-231 cells,and stable cell lines were obtained after puromycin selection.The knockdown efficiency and cell proliferation effect were determined in stable cell lines treated with doxycycline(DOX)by Western blot and MTT assays respectively.Results:The pLKO.1-tet-on-shPICH-Puromycin plasmids were correctly constructed.The cell lines stably expressing inducible shRNA targeting PICH were established.The PICH expression was reduced after DOX treatment and PICH knockdown significantly suppressed proliferation of MDA-MB-231 cells.Conclusion:The stable inducible PICH knockdown MDA-MB-231 cell line was successfully established,laying a foundation for further study of PICH function in triple-negative breast cancer.

关 键 词：polo样激酶1相关检查点解旋酶(PICH) 三阴乳腺癌 可诱导稳定敲低 细胞增殖 

分 类 号：Q78[生物学—分子生物学]