摘要
目的探讨上调micro RNA-206(mi R-206)和micro RNA-1(mi R-1)表达对乳腺癌干细胞增殖的影响以及其作用机制。方法采用流式细胞分选技术从乳腺癌细胞株MCF-7中分离出乳腺癌干细胞。实验分为空白对照组、阴性对照组、mi R-206转染组、mi R-1转染组。除空白对照组外,其他各组分别转染阴性对照mimic、hsa-mi R-206 mimic、hsa-mi R-1 mimic。应用实时定量PCR检测mi R-206、mi R-1以及转录因子EVI-1基因的表达水平;Western blot法检测EVI-1蛋白的表达;应用MTT法检测mi R-206、mi R-1对乳腺癌干细胞增殖能力的影响。结果从MCF-7细胞系中分选出CD44+/CD24-/low乳腺癌干细胞,经无血清培养后可以成功传代,用于后续试验;转染hsa-mi R-206 mimic、hsa-mi R-1 mimic 48 h后mi R-206、mi R-1相对表达水平提高,EVI-1m RNA表达水平明显下降;Western blot、MTT结果显示,上调mi R-206和mi R-1表达水平后显著降低EVI-1蛋白的表达,抑制了乳腺癌干细胞增殖能力。乳腺癌干细胞中mi R-206、mi R-1表达水平以及EVI-1蛋白表达水平的差异有统计学意义(P<0.05)。结论上调mi R-206和mi R-1表达能够抑制乳腺癌干细胞增殖能力,其机制可能与下调EVI-1的表达有关。
Objective To investigate the effects of up-regulated miR-206/miR-1 on the proliferation of breast cancer stem cells and the effect mech-anism. Methods Breast cancer stem cells（BCSCs）were isolated from breast cancer cell line MCF-7 by fluorescence-activated cell sorting. Cells in the experiment were divided into the blank control group,the negative control group,the miR-206 group and the miR-1 group. The BCSCs were transfected by negative control mimic,hsa-miR-206mimic and hsa-miR-1mimic in all groups except the blank control group. MiR-206and miR-1 expression levels as well as the transcription factor EVI-1 gene were detected by real time PCR. The expression levels of the transcription factor EVI-1 protein were detected by Western blot. MTT method was used to detect the effects of miR-206 and miR-1 on the proliferation of BCSCs. Results The BCSCs（CD44＋/CD24-/low cells）isolated from MCF-7 cell lines were successfully cultured in serum-free medium for subsequent studies. After transfection of hsa-miR-206mimic and hsa-miR-1mimic for 48 hours,miR-206and miR-1relative expression levels increased. EVI-1mRNA ex-pression levels significantly decreased. The results of Western blot and MTT showed that up-regulated expression levels of miR-206 and miR-1 could significantly reduce the expression of EVI-1 protein and inhibited the proliferation of BCSCs. The differences in levels of miR-206,miR-1 and EVI-1 protein were statistically significant（P〈0.05）. Conclusion Up-regulated miR-206 and miR-1 expression can inhibit the proliferation ability of BCSCs,which may be related to the down-regulation of EVI-1.
出处
《中国医科大学学报》
CAS
CSCD
北大核心
2015年第5期394-399,共6页
Journal of China Medical University
基金
国家自然科学基金(81372812)
