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

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2.0μg/m L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。

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-10 A-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 2.0μg/m L 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-10 A-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.