HOXD3在乳腺癌细胞干性和化疗耐药性中的作用及机制研究

摘要

目的:探讨HOXD3表达对乳腺癌细胞干性的影响,并研究HOXD3表达与乳腺癌细胞化疗耐药的关系.方法:收集2006年1月至2008年12月哈尔滨医科大学附属肿瘤医院87例乳腺癌患者组织标本.采用免疫组织化学染色法检测乳腺癌细胞和组织中HOXD3表达;采用RT-PCR、Western blot和免疫荧光染色法检测HOXD3在顺铂或阿霉素耐药细胞系MDA-MB-231和MDA-MB-435中的表达水平,分析HOXD3过表达对乳腺癌细胞系MDA-MB-231和MDA-MB-435的干细胞生物标志物表达水平的影响;采用MTT法和集落形成实验分析HOXD3在乳腺癌细胞化疗耐药中的作用.结果:乳腺癌组织中HOXD3 mRNA相对表达量显著高于癌旁正常组织,乳腺癌细胞系MDA-MB-231、MDA-MB-435和MCF-7的HOXD3 mRNA相对表达量均高于正常乳腺上皮细胞系MCF-10A(均P＜0.05).顺铂或阿霉素耐药的细胞系MDA-MB-231和MDA-MB-435的半抑制浓度(half maximal inhibi-tory concentration,IC50)分别为(20.82±0.05)μmol/L和(19.69±0.47)μmol/L,或(32.26±0.23)mmol/L和(26.08±0.55)mmol/L,均高于对应原始细胞系(均P＜0.05);耐药倍数分别为2.47和3.10倍,或1.86和2.08倍.HOXD3过表达MDA-MB-231、MDA-MB-435的肿瘤球体数目、干细胞生物标志物的表达水平均明显增加(均P＜0.05).结论:HOXD3过表达对乳腺癌细胞干性的维持及化疗耐药性的发生发挥重要的作用,为制定针对肿瘤干细胞的分子靶向治疗提供理论参考.%Objective: To investigate the effect of HOXD3 expression on the stem cell-like characteristics of breast cancer cells and the relationship between HOXD3 expression and multi-drug resistance in breast cancer cells. Methods: From January 2006 to December 2008, 87 specimens of breast cancer patients from the Affiliated Tumor Hospital of Harbin Medical University were collected. The ex-pression of HOXD3 in breast cancer cells and tissues was detected by immunohistochemical staining method. The expression levels of HOXD3 in CDDP or DOX-resistant cell lines MDA-MB-231 and MDA-MB-435 were detected by RT-PCR, Western blot and immunofluo-rescence staining. The effect of HOXD3 overexpression on the expression levels of stem cell biomarkers in breast cancer cell lines MDA-MB-231 and MDA-MB-435 was analyzed. MTT assay and colony formation assay were used to demonstrate the role of HOXD3 in che-motherapy resistance of breast cancer cells. Results: The relative expression of HOXD3 mRNA in breast cancer was 4.16, which was sig-nificantly higher than 2.05 in normal tissues adjacent to cancer; the relative expression levels of HOXD3 mRNA in breast cancer cell lines MDA-MB-231, MDA-MBB-435 and MCF-7 were 3.25, 2.84 and 2.23, which were all higher than 1.00 in normal breast epithelial cell line MCF-10A ( all P＜0.05 ). The IC50s of MDA-MB-231 and MDA-MB-435 cell lines resistant to CDDP or DOX were (20.82±0.05) μmol/L, (19.69±0.47) μmol/L, (32.26±0.23) mmol/L and (26.08±0.55) mmol/L, respectively. Both were higher than the corresponding original cell lines (all P＜0.05), and the drug resistance times were 2.47 and 3.10 or 1.86 and 2.08, respectively. The number of tumor spheres and stem cell biomarker expression levels of MDA-MB-231 and MDA-MB-435 with HOXD3 overexpression were significantly in-creased (all P＜0.05). Conclusions: The expression of HOXD3 plays an important role in the maintenance of stem cell-like properties and drug resistance of breast cancer cells. The results of this study will help us better understand the complexity of breast cancer and pro-vide a theoretical basis for the development of targeted molecular therapy.