文章摘要
程 腾,杜雅莹,张磐石,胡晓鹏,夏文飞.miR-429对乳腺癌干细胞干性维持和体内成瘤能力的影响[J].,2019,19(13):2429-2433
miR-429对乳腺癌干细胞干性维持和体内成瘤能力的影响
Effects of miR-429 on Stemness Maintenance and Tumorigenesis in Vivo of Breast Cancer
投稿时间:2018-11-14  修订日期:2018-12-10
DOI:10.13241/j.cnki.pmb.2019.13.006
中文关键词: 乳腺癌  干细胞  成球能力  CD44+CD24-表型细胞亚群  体内成瘤
英文关键词: Breast cancer  Stem cells  Mammosphere forming ability  CD44+CD24-phenotype cell subsets  Tumorigenesis in vivo
基金项目:湖北省科技计划基金项目(2014CA1379)
作者单位E-mail
程 腾 华中科技大学同济医学院附属同济医院普外科 湖北 武汉 430030 doctorcheng888@126.com 
杜雅莹 华中科技大学同济医学院附属同济医院普外科 湖北 武汉 430030  
张磐石 华中科技大学同济医学院附属同济医院普外科 湖北 武汉 430030  
胡晓鹏 华中科技大学同济医学院附属同济医院普外科 湖北 武汉 430030  
夏文飞 华中科技大学同济医学院附属同济医院普外科 湖北 武汉 430030  
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中文摘要:
      摘要 目的:探究miR-429在乳腺癌干性维持中所发挥的作用,并探索miR-429对乳腺癌干细胞体内成瘤能力的影响。方法:无血清悬浮培养法用于培养经流式细胞仪分选得到的CD44+CD24-表型乳腺癌细胞系干细胞MCF-7-S、SKBR3-S、MDA-MB-231-S及乳腺正常上皮干细胞MCF-10A-S,实时荧光定量聚合酶链式反应(qRT-PCR)用于检测miR-429在上述4株干细胞中的表达。将包含miR-429的重组慢病毒质粒及其阴性对照空载体质粒vector分别以病毒:细胞数量为15:1的比例感染MDA-MB-231细胞,经2.0 μg/mL嘌呤霉素筛选,成功构建稳定表达miR-429或vector的MDA-MB-231细胞,经流式分选出上述两株稳转细胞株的CD44+CD24-表型干细胞MDA-MB-231-Svector和MDA-MB-231-SmiR-429。无血清悬浮培养后,镜下观察过表达miR-429对肿瘤球形成能力的影响,流式细胞术检测过表达miR-429对CD44+CD24-表型细胞亚群比例的影响,Western Blot检测过表达miR-429对乳腺癌干细胞干性相关因子ALDH1、SOX2和Bmi1蛋白表达的影响,将MDA-MB-231-Svector和MDA-MB-231-SmiR-429干细胞分别注射到BALB/c裸鼠右侧胸壁第二对乳腺脂肪垫中,构建乳腺癌干细胞裸鼠移植瘤模型,观察过表达miR-429对裸鼠体内成瘤能力的影响。结果:与MCF-10A-S相比,miR-429在MCF-7-S、SKBR3-S和MDA-MB-231-S细胞系中的表达水平均异常降低,其中,miR-429在MDA-MB-231-S细胞中表达最低(P<0.05)。与MDA-MB-231-Svector细胞相比,经流式分选后的CD44+CD24-表型MDA-MB-231-SmiR-429干细胞形成的肿瘤球的大小和数量、分选时CD44+CD24-表型细胞亚群的比例、ALDH1、SOX2和Bmi1的蛋白表达水平以及裸鼠体内成瘤的体积和重量均显著降低(P<0.05)。结论:miR-429可降低乳腺癌干细胞的干性和体内成瘤能力,其可能是抑制乳腺癌转移和耐药的关键分子。
英文摘要:
      ABSTRACT Objective: To explore the role of miR-429 in the stemness maintenance of breast cancer and explore the effect of miR-429 on tumorigenic ability in vivo of breast cancer stem cells. Methods: The CD44+CD24- phenotype breast cancer stem cells MCF-7-S, SKBR3-S, MDA-MB-231-S and normal mammary gland stem cells MCF-10A-S sorted by Flow Cytometry were cultured in serum-free suspension culture. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-429 in the above four stem cells. The recombinant lentiviral vector containing miR-429 and its negative control vector were respectively transfected into MDA-MB-231 cells in the ratio of virus/cell number with 15:1, and screened with 2.0 μg/mL puromycin to construct stable expressing miR-429 and vector MDA-MB-231 cells, CD44+CD24- phenotype breast cancer stem cells MDA-MB-231-Svector and MDA-MB-231-SmiR-429 were sorted by Flow Cytometry from the above two cells, then the cells were serum-free suspension cultured. In order to assess the effects of miR-429 overexpression in MDA-MB-231 cells, Microscope was used to observe of the mammosphere forming ability, Flow cytometry was used to detect of the percentage of CD44+CD24- phenotype cell subsets, Western Blot was used to detect the expression of ALDH1, SOX2 and Bmi1 proteins expression. MDA-MB-231-Svector and MDA-MB-231-SmiR-429 cells were respectively injected into the second pair of breast pat pad on the right side of the chest in BALB/c nude mice to construct the transplanted tumor model of breast cancer stem cells in nude mice. The effects of overexpression miR-429 on the tumorigenic ability in nude mice were observed. Results: The expression of miR-429 in MCF-7-S, SKBR3-S and MDA-MB-231-S was significantly lower than that of MCF-10A-S, which was lowest in MDA-MB-231-S(P<0.05). The size and number of tumor mammospheres, the proportion of CD44+CD24- phenotype cell subsets after sorting, the protein expression levels of ALDH1, SOX2 and Bmi1, the tumor volume and weight in nude from MDA-MB-231-SmiR-429 cells were both significantly less than or lower than that of MDA-MB-231-Svector cells(P<0.05). Conclusion: miR-429 can decrease the stemness and tumorigenicity of breast cancer stem cells and may be a key molecule in breast cancer metastasis and drug resistance.
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