【摘要】：目的:建立穩(wěn)定表達可誘導型polo樣激酶1(polo-like kinase 1,PLK1)小發(fā)夾RNA(small hairpin RNA,shRNA)的乳腺癌MDA-MB-231細胞系并研究PLK1敲低對MDA-MB-231細胞增殖、細胞周期的影響并探討其可能的機制。方法:根據(jù)si RNA的原理設(shè)計2對靶向PLK1的shRNA序列,構(gòu)建慢病毒表達質(zhì)粒(PLK1 shRNA重組質(zhì)粒);先用p LV-t TR/KRAB-Red空載體制備慢病毒并感染MDA-MB-231細胞;在此基礎(chǔ)上再用PLK1 shRNA重組質(zhì)粒制備慢病毒并感染上述MDA-MB-231細胞;對上述2次慢病毒感染的MDA-MB-231細胞用強力霉素(doxycycline,DOX)處理96 h后,用q RT-PCR及Western blot檢測PLK1m RNA及蛋白的表達;用MTT法檢測細胞增殖;用流式細胞術(shù)及碘化吡啶(PI)染色檢測細胞周期。結(jié)果:成功構(gòu)建靶向PLK1的shRNA慢病毒表達質(zhì)粒,包裝出慢病毒并感染MDA-MB-231細胞;成功建立穩(wěn)定表達可誘導型PLK1 shRNA的MDA-MB-231細胞系,通過DOX誘導,可明顯抑制該細胞系中PLK1在m RNA及蛋白水平的表達(P0.01);PLK1敲低可明顯抑制MDA-MB-231細胞的增殖(P0.05);流式細胞術(shù)檢測發(fā)現(xiàn)PLK1敲低可阻滯MDA-MB-231細胞于G2/M期(P0.05)。結(jié)論:DOX誘導的PLK1敲低可明顯抑制MDA-MB-231細胞增殖并阻滯細胞于G2/M期;經(jīng)PLK1介導的腫瘤生物學行為變化,其機制可能涉及ERK1/2/Fra1/ZEB1信號通路。
[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.
【作者單位】： 三峽大學醫(yī)學院;
【基金】：國家自然科學基金資助項目(編號:81550029、30800410) 湖北省自然科學基金資助項目(編號:2015CFB198) 三峽大學人才啟動基金資助項目(編號:KJ2014B064) 腫瘤微環(huán)境與免疫治療湖北省重點實驗室(三峽大學)開放基金資助項目(編號:2015KZL02) 宜昌市科學技術(shù)局資助項目(編號:A16-301-31)
【分類號】：R737.9
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本文編號：2211648