三阴性乳腺癌MDA-MB-231细胞低氧模型的建立及作用研究

Impact of different hypoxia conditions on the MDA-MB-231 triple-negative human breast cancer cells

目的:研究不同的低氧环境对三阴性乳腺癌细胞株MDA-MB-231增殖和侵袭迁移能力的影响。方法:将MDA-MB-231细胞分为间歇低氧组、持续低氧组和常氧组,采用划痕法和Transwell小室法进行迁移实验;CCK8法检测细胞增殖;荧光定量PCR和Western blot分别检测低氧诱导因子-1α(HIF-1α)和波形蛋白(vimentin)的m RNA和蛋白表达;si RNA干扰间歇低氧组细胞HIF-1α基因表达,在此基础上进一步检测低氧细胞的迁移、增殖及vimentin蛋白改变。结果:间歇低氧促进MDA-MB-231细胞迁移,且其促进作用显著高于持续低氧组(P<0.05);间歇低氧和持续低氧均抑制细胞增殖,但持续低氧对细胞增殖抑制的作用在低氧48 h后才较为明显(P<0.05);间歇性低氧组细胞HIF-1α和vimentin蛋白表达均显著高于持续低氧细胞。si RNA干扰间歇低氧组细胞HIF-1α表达后,其vimentin蛋白下调,细胞迁移能力下降(P<0.05),但增殖水平未受到明显影响(P>0.05)。结论:间歇低氧可诱导高侵袭表型的MDA-MB-231细胞株,其侵袭力增强与HIF-1α表达增加进而上调vimentin有关。

OBJECTIVE：To study the influence of hypoxia on metastatic potential of the MDA-MB-231 triplenegative human breast cancer cells.METHODS：MDA-MB-231 cells were divided into three hypoxia groups：intermittent hypoxia（IH）,continuous hypoxia（CH） and normoxic（N）.Cell migration and invasion ability were analyzed by wound healing and the Boyden chamber assay.Cell proliferation was analyzed by the CCK-8 assay.Hypoxia-inducible factor-la（HIF-1α）and vimentin expression in response to different hypoxic environments were analyzed by Western blot and real-time PCR assays.HIF-1α expression via siRNA knockout was investigated.RESULTS：IH-treated cells exhibited higher invasiveness than the CH-treated cells（P〈0.05）.On the other hand,IH significantly inhibited cell proliferation while CH did not show such effect until 48 h later（P〈0.05）.IH induced a greater effect on HIF-1α protein accumulation and vimentin upregulation.Knockdown of HIF-1α by siRNA abolished IH-induced cell migration and vimentin upregulation（P〈0.05）.However,knockdown of HIF-1α had no effect on proliferation of MDA-MB-231 cells（P〉0.05）.CONCLUSION：IH had a more pronounced effect on enhancing the invasive phenotype of MDA-MB-231 cells than CH,and HIF-1α activation together with increased vimentin upregulation might be responsible for the phenotypic change.