Effect of silencing HIF-2α expression on the enrichment of breast cancer stem cell microspheres under hypoxia
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摘要:
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 cancerstem cells, BCSCs)微球体富集的影响。 方法 构建HIF-2αRNA干扰慢病毒载体, 并转染至MCF-7细胞, RT-PCR和Western blot检测HIF-2αmRNA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-2αmRNA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。 Abstract: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-2 α gene was constructed and transfected into Michigan Cancer Foundation(MCF)-7 cells.The mRNAand 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-2 α may be a new candidate gene for BCSC-targeting therapy. -
图 1 PCR鉴定HIF-2α基因RNA干扰慢病毒载体的阳性克隆
Figure 1. Positive clones of lentivirus vector harboring RNAi targeting HIF-2αgene identified by PCR
1 and 2:Negative clone group(289 bp); 3 and 4:Positive clone group(343 bp); 5 and 6:Empty vector group(289 bp); 7:Negative control group(ddH2O); M: Marker
图 2 RT-PCR和Western blot检测各组MCF-7细胞中HIF-2 α mRNA及蛋白表达
Figure 2. HIF-2α mRNA and protein expressions in MCF-7 cells from different groups, as detected by RT-PCR and Western blot, respectively (x±s, n=3)
1:RNA interference group(20% O2); 2:RNA interference group(1% O2)3:Empty vector group(20% O2); 4:Empty vector group(1% O2); M: Marker
图 3 MTT法检测各组MCF-7细胞增殖抑制率
Figure 3. Proliferation inhibition rate of MCF-7 cells in different groups as determined by the MTT assay
图 4 荧光显微镜下观察低氧下各组MCF-7细胞微球体形成情况
Figure 4. Microsphere formation of MCF-7 cells for different groups un-der hypoxia, as observed by fluorescent microscopy
A: Non-transfected group(×40);B: Empty vector group(×40);C: RNA interference group(×40);D: Non-transfected group(×200);E: Empty vector group(×200);F: RNA interference group(×200)
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