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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。
Objective: To investigate the expression of let-7 in breast cancer stem cells and to explore the characteristics and mechanism of let-7 in breast cancer stem cells. Methods: The SP and NSP subsets of breast cancer cell line MCF-7 were sorted by SP method. The expression of let-7a / b / c of SP and NSP in MCF-7 cell line was detected by real-time quantitative PCR The expression of Ras and ERK protein in the SP and NSP subgroups of MCF-7 cell line was detected by Western blot, and the mechanism of let-7 in maintaining the characteristics of breast cancer stem cells was explored. RESULTS: SP group cells accounted for 3.3% of MCF-7 cells. After adding verapamil to Hoechst33342 cells, the ratio of SP group to MCF-7 cells decreased to 0.4%. The expression of let-7miRNA in SP cells was lower than that in NSP cells, and the expression of let-7b / c was the highest in SP and NSP subgroups. The expression of p-Ras and p-ERK in SP cells was higher than that in NSP cells, t -Ras and t-ERK expression no significant difference. Conclusion: SP method is an effective method for the isolation of stem cells. There is a subset of cancer stem cells in breast cancer cell line MCF-7. The expression of let-7 in breast cancer stem cells is lower than that in normal tumor cells. However, p-Ras, p -ERK expression in breast cancer stem cells than normal tumor cells, let-7 low expression of the loss of Ras mRNA inhibition, the Ras signal transduction pathway activation, and phosphorylation of p-Ras and p-ERK expression High, maintain the function of breast cancer stem cells.