CAJ | 학술논문

目的:探讨沉默低氧诱导因子-2α(hypoxia-inducible factor-2α,HIF-2α)表达对低氧下乳腺癌干细胞(breast cancer stem cells,BCSCs)微球体富集的影响.方法:构建HIF-2α RNA干扰慢病毒载体,并转染至MCF-7细胞,RT-PCR和Western blot检测HIF-2α mRNA及蛋白表达;MTT检测MCF-7细胞在不同氧浓度下增殖活性;显微镜下观察低氧下BCSCs微球体形成情况;有限稀释法检测微球体细胞单克隆形成能力;RT-PCR检测微球体细胞干细胞相关标志物ABCG2、CD44及OCT-4 mRNA表达.结果:成功构建了HIF-2α基因siRNA慢病毒表达载体,RT-PCR和Western blot结果显示干扰组细胞HIF-2α mRNA及蛋白表达均明显降低(P<0.05).与未转染组及空载体组比较,低氧下 RNA 干扰组细胞增殖活性及 BCSCs 单克隆形成能力明显降低(P<0.05);RT-PCR结果亦显示RNA干扰组BCSCs相关标志物ABCG2、CD44及OCT-4 mRNA表达显著降低(P<0.05).结论:低氧能够诱导和强化MCF-7细胞乳腺癌癌干细胞样特性,而沉默HIF-2α表达可抑制低氧下BCSCs富集,提示HIF-2α有望成为乳腺癌治疗新的靶点.
목적:탐토침묵저양유도인자-2α(hypoxia-inducible factor-2α,HIF-2α)표체대저양하유선암간세포(breast cancer stem cells,BCSCs)미구체부집적영향.방법:구건HIF-2α RNA간우만병독재체,병전염지MCF-7세포,RT-PCR화Western blot검측HIF-2α mRNA급단백표체;MTT검측MCF-7세포재불동양농도하증식활성;현미경하관찰저양하BCSCs미구체형성정황;유한희석법검측미구체세포단극륭형성능력;RT-PCR검측미구체세포간세포상관표지물ABCG2、CD44급OCT-4 mRNA표체.결과:성공구건료HIF-2α기인siRNA만병독표체재체,RT-PCR화Western blot결과현시간우조세포HIF-2α mRNA급단백표체균명현강저(P<0.05).여미전염조급공재체조비교,저양하 RNA 간우조세포증식활성급 BCSCs 단극륭형성능력명현강저(P<0.05);RT-PCR결과역현시RNA간우조BCSCs상관표지물ABCG2、CD44급OCT-4 mRNA표체현저강저(P<0.05).결론:저양능구유도화강화MCF-7세포유선암암간세포양특성,이침묵HIF-2α표체가억제저양하BCSCs부집,제시HIF-2α유망성위유선암치료신적파점.
Objective: This work aims to explore the effect of silencing hypoxia-inducible factor (HIF)-2α expression on the enrichment of breast cancer stem cell (BCSC) microspheres under hypoxia. Methods: A lentiviral vector of the RNA interference (RNAi) of human HIF-2α gene was constructed and transfected into Michigan Cancer Foundation (MCF)-7 cells. The mRNA and protein expressions of HIF-2α were determined using real time-polymerase chain reaction (RT-PCR) and Western blot, respectively. The proliferation activity of the MCF-7 cells under different oxygen concentrations was measured using the methyl thiazolyl tetrazolium assay. The formation of the BCSC microspheres under hypoxia was observed by a fluorescence microscope. The monoclonal formation ability of the microsphere cells was detected by limiting dilution assay. The mRNA expressions of the BCSC markers, ABCG, CD44, and OCT-4, were tested by RT-PCR. Results: The siRNA expression vector targeting the HIF-2α gene was successfully constructed. Both the HIF-2a mRNA and protein levels noticeably decreased in the MCF-7 cells transfected with the RNAi expression vector (P<0.05). Compared with the groups with empty vectors or those that were not transfected, the proliferation activity of MCF-7 cells and the monoclonal formation ability of BCSCs in the RNAi group were markedly inhibited after silencing the HIF-2α gene (P<0.05). RT-PCR results showed that the mRNA expressions of BCSC markers ABCG2, CD44, and OCT-4 in cells from the RNAi group decreased significantly under hypoxia (P<0.05). Conclusion: Hypoxia induced and enhanced cancer stem cell-like characteristics in MCF-7 cells. HIF-2α silencing leads to the decreased enrichment of BCSC microspheres under hypoxia. HIF-2α may be a new candidate gene for BCSC-targeting therapy.