可诱导型PLK1 shRNA的乳腺癌MDA-MB-231细胞系的建立及PLK1敲低对细胞增殖的影响  

Establishment of MDA-MB-231 cell line stably expressing inducible sh RNA targeting PLK1 and effects of PLK1 knockdown on cell proliferation of MDA-MB-231 cells

作　　者：薛慧[1] 郭欣[1] 李娜[1] 李志红[1] 彭帆[1] 肖方祥[1] 袁成福[1] 

机构地区：[1]三峡大学医学院,宜昌443002

出　　处：《重庆医科大学学报》2017年第11期1400-1405,共6页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81550029;30800410);湖北省自然科学基金资助项目(编号:2015CFB198);三峡大学人才启动基金资助项目(编号:KJ2014B064);肿瘤微环境与免疫治疗湖北省重点实验室(三峡大学)开放基金资助项目(编号:2015KZL02);宜昌市科学技术局资助项目(编号:A16-301-31)

摘　　要：目的:建立稳定表达可诱导型polo样激酶1(polo-like kinase 1,PLK1)小发夹RNA(small hairpin RNA,shRNA)的乳腺癌MDA-MB-231细胞系并研究PLK1敲低对MDA-MB-231细胞增殖、细胞周期的影响并探讨其可能的机制。方法:根据si RNA的原理设计2对靶向PLK1的shRNA序列,构建慢病毒表达质粒(PLK1 shRNA重组质粒);先用p LV-t TR/KRAB-Red空载体制备慢病毒并感染MDA-MB-231细胞;在此基础上再用PLK1 shRNA重组质粒制备慢病毒并感染上述MDA-MB-231细胞;对上述2次慢病毒感染的MDA-MB-231细胞用强力霉素(doxycycline,DOX)处理96 h后,用q RT-PCR及Western blot检测PLK1m RNA及蛋白的表达;用MTT法检测细胞增殖;用流式细胞术及碘化吡啶(PI)染色检测细胞周期。结果:成功构建靶向PLK1的shRNA慢病毒表达质粒,包装出慢病毒并感染MDA-MB-231细胞;成功建立稳定表达可诱导型PLK1 shRNA的MDA-MB-231细胞系,通过DOX诱导,可明显抑制该细胞系中PLK1在m RNA及蛋白水平的表达(P<0.01);PLK1敲低可明显抑制MDA-MB-231细胞的增殖(P<0.05);流式细胞术检测发现PLK1敲低可阻滞MDA-MB-231细胞于G2/M期(P<0.05)。结论:DOX诱导的PLK1敲低可明显抑制MDA-MB-231细胞增殖并阻滞细胞于G2/M期;经PLK1介导的肿瘤生物学行为变化,其机制可能涉及ERK1/2/Fra1/ZEB1信号通路。Objective ：To establish MDA-MB-231 cell line stably expressing inducible small hairpin RNA（shRNA） targeting polo-like kinase 1 （PLK1）,to explore the effects of PLK1 knockdown on cell proliferation and Cell cycle in MDA-MB-231 cells as well as to explore its possible mechanism. Methods ：Two oligonucleotides targeting PLK1 were synthesized and cloned into lentivirus expression plasmid pLVTHM according to siRNA principle（named PLK1 shRNA）. MDA-MB-231 cells were infected at first with lentivirus pre- pared by pLV-tTR/KRAB-Red empty vector, and then infected with another lentivirus prepared by PLK1 shRNA recombinant plasmid. MDA-MB-231 cells infected with aforementioned lentivirus were treated with doxycycline（DOX） for 96 hours,the expression of PLK1mRNA and protein was determined by real time PCR（qRT-PCR） and Western blot respectively. MTT method was used to deter- mine cell proliferation and flow cytometry and PI staining were used to detect cell cycle. Results：The lentivirus expression plasmids containing PLK1 shRNA were constructed successfully, and MDA-MB-231 cells were infected with the lentivirus pre- pared by the aforementioned plasmids. MDA-MB-231 cell line expressing stably inducible shRNA targeting PLK1 was estab- lished successfully and PLK1 expression levels were decreased obviously at mRNA and protein levels after induction by DOX （P〈0.01）. PLK1 knockdown could obviously inhibit the prolif-eration of MDA-MB-231 cells and arrest the cell cycle in G2/M phase（P〈0.05）. Conclusions：PLK1 knockdown induced by DOX could inhibit MDA-MB-231 cell proliferation and arrest cell cycle in G2/M phase,its possible mechanism may involve in ERK1/2/ Fral/ZEB1 signal pathway.

关 键 词：POLO样激酶1 可诱导型shRNA 乳腺癌 

分 类 号：R737.9[医药卫生—肿瘤]