Let-7维持乳腺癌干细胞的特性及机制的研究

Role of let-7 in maintaining characteristics of breast cancer stem cells

目的:研究let-7在乳腺癌干细胞中的表达,探索let-7影响乳腺癌干细胞的特性及机制。方法:采用SP分选法,分选乳腺癌细胞系MCF-7中的SP及NSP细胞亚群;运用实时定量PCR法检测MCF-7细胞系中SP、NSP亚群let-7a/b/c的表达;通过Western blot法检测Ras、ERK蛋白在MCF-7细胞系中SP、NSP亚群中的表达,探索let-7维持乳腺癌干细胞特性的机制。结果:SP侧群细胞占MCF-7细胞的3.3%,加入维拉帕米抑制干细胞外排荧光染料Hoechst33342的功能后,SP侧群细胞占乳腺癌MCF-7细胞的比例下降至0.4%。Let-7miRNA在SP细胞中的表达低于NSP细胞,其中let-7b/c在SP及NSP亚群中表达差异最大;p-Ras、p-ERK在SP细胞中的表达高于NSP细胞,t-Ras及t-ERK的表达无明显差异。结论:SP法是一种可有效分离干细胞的方法,乳腺癌细胞系MCF-7中存在肿瘤干细胞亚群;let-7在乳腺癌干细胞中的表达低于普通肿瘤细胞,而p-Ras、p-ERK在乳腺癌干细胞中的表达高于普通肿瘤细胞,let-7的低表达失去了对Ras mRNA的抑制,使Ras信号转导通路激活,进而磷酸化的p-Ras和p-ERK表达升高,维持乳腺癌干细胞的功能。

AIM： To observe the expression of let-7 in breast cancer stem cells and explore the role of let-7 in maintaining the characteristics of breast cancer stem cells.METHODS： We separated breast cancer stem cells（SP and NSP） from MCF-7 cell line using SP sorting,and observed the expression of let-7a/b/c on SP and NSP cells using quantitative real-time PCR and the expressions of Ras and ERK using Western blotting to study the mechanism by which let-7 maintains the characteristics of breast cancer stem cells.RESULTS： The SP cells accounted for 3.3% in MCF-7 cells,however,the rate droped to 0.4% when verapamil was added into the process of seperation.The level of Let-7a/b/c in SP cells were lower than that in NSP cells,and among let-7 miRNAs,let-7b/c showed the most obvious difference.The expressions of t-Ras and t-ERK showed no difference between SP and NSP cells,nevertheless,the expressions of p-Ras,p-ERK were higher in SP cells than in NSP cells.CONCLUSION： SP sorting is an effective method to separate cancer stem cells.There do exist cancer stem cells in MCF-7 breast cancer cell line.Let-7 is down-regulated in SP cells,and the down-regulation makes let-7 lose the opportunity to restrain Ras mRNA,finally,p-Ras and p-ERK are activated.They play an important role in maintaining the characteristics of breast cancer stem cells.