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
发布时间：2018-08-29 15:39
【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(P0.01);PLK1敲低可明显抑制MDA-MB-231细胞的增殖(P0.05);流式细胞术检测发现PLK1敲低可阻滞MDA-MB-231细胞于G2/M期(P0.05)。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。
[Abstract]:Aim: to establish a breast cancer MDA-MB-231 cell line stably expressing inducible polo like kinase 1 (polo-like kinase 1 / PLK1) with small hairpin RNA (small hairpin RNA,shRNA and to investigate the effect of PLK1 knockout on the proliferation and cell cycle of MDA-MB-231 cells and to explore its possible mechanism. Methods: according to the principle of si RNA, two pairs of shRNA sequences targeting PLK1 were designed to construct the lentivirus expression plasmid (PLK1 shRNA recombinant plasmid), and the lentivirus was first prepared by pLV-t TR/KRAB-Red empty vector and infected with MDA-MB-231 cells. On this basis, lentivirus was prepared with PLK1 shRNA recombinant plasmid and infected with MDA-MB-231 cells, and the expression of PLK1m RNA and protein was detected by Q RT-PCR and Western blot after MDA-MB-231 cells were treated with doxycycline,DOX for 96 h. Cell proliferation was detected by MTT assay, cell cycle was detected by flow cytometry and (PI) staining of pyridine iodide. Results: the shRNA lentivirus expression plasmid targeting PLK1 was successfully constructed, the lentivirus was packaged and infected with MDA-MB-231 cells, and the MDA-MB-231 cell line expressing inducible PLK1 shRNA was successfully established, which was induced by DOX. The expression of PLK1 in m RNA and protein level (P0.01) could significantly inhibit the proliferation of MDA-MB-231 cells (P0.05). Flow cytometry showed that PLK1 knockout could block the G _ 2 / M phase of MDA-MB-231 cells (P0.05). Conclusion PLK1 knockout induced by PLK1 can significantly inhibit the proliferation of MDA-MB-231 cells and block the proliferation of MDA-MB-231 cells in G _ 2 / M phase, and the mechanism of ERK1/2/Fra1/ZEB1 signaling pathway may be involved in the changes of tumor biological behavior mediated by PLK1.
【作者单位】： 三峡大学医学院;
【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)
【分类号】：R737.9
，
本文编号：2211648
[Abstract]:Aim: to establish a breast cancer MDA-MB-231 cell line stably expressing inducible polo like kinase 1 (polo-like kinase 1 / PLK1) with small hairpin RNA (small hairpin RNA,shRNA and to investigate the effect of PLK1 knockout on the proliferation and cell cycle of MDA-MB-231 cells and to explore its possible mechanism. Methods: according to the principle of si RNA, two pairs of shRNA sequences targeting PLK1 were designed to construct the lentivirus expression plasmid (PLK1 shRNA recombinant plasmid), and the lentivirus was first prepared by pLV-t TR/KRAB-Red empty vector and infected with MDA-MB-231 cells. On this basis, lentivirus was prepared with PLK1 shRNA recombinant plasmid and infected with MDA-MB-231 cells, and the expression of PLK1m RNA and protein was detected by Q RT-PCR and Western blot after MDA-MB-231 cells were treated with doxycycline,DOX for 96 h. Cell proliferation was detected by MTT assay, cell cycle was detected by flow cytometry and (PI) staining of pyridine iodide. Results: the shRNA lentivirus expression plasmid targeting PLK1 was successfully constructed, the lentivirus was packaged and infected with MDA-MB-231 cells, and the MDA-MB-231 cell line expressing inducible PLK1 shRNA was successfully established, which was induced by DOX. The expression of PLK1 in m RNA and protein level (P0.01) could significantly inhibit the proliferation of MDA-MB-231 cells (P0.05). Flow cytometry showed that PLK1 knockout could block the G _ 2 / M phase of MDA-MB-231 cells (P0.05). Conclusion PLK1 knockout induced by PLK1 can significantly inhibit the proliferation of MDA-MB-231 cells and block the proliferation of MDA-MB-231 cells in G _ 2 / M phase, and the mechanism of ERK1/2/Fra1/ZEB1 signaling pathway may be involved in the changes of tumor biological behavior mediated by PLK1.
【作者单位】： 三峡大学医学院;
【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)
【分类号】：R737.9
，
本文编号：2211648
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