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目的探讨沉默乳腺癌MCF-7细胞B细胞特异的莫洛尼鼠白血病病毒插入位点1(B-cell-specific moloney murine leukemia virusinsertion site 1,Bmi-1)基因表达对乳腺癌干细胞生物学特性的影响。方法构建Bmi-1基因si RNA逆转录病毒表达载体,转染MCF-7细胞,筛选转染稳定细胞株,确定转染细胞组;设立干扰序列载体转染细胞组(阴性对照组)及空白对照组。无血清-有血清交替悬浮培养法富集乳腺癌干细胞,制备单细胞悬液。Western-blot检测乳腺癌干细胞Bmi-1蛋白表达。RT-PCR检测各组干细胞中人端粒酶逆转录酶(human telomerase revere transcriptase,h TERT)、血管内皮生长因子C(vascular endothelial growth factor VEGF-C)mRNA的表达。采用单因素方差分析对各组进行对比分析,P<0.05为差异有统计学意义。结果转染组h TERT、VEGF-C mRNA表达为(3.46±0.50)、(2.98±0.18)与空白对照[(6.88±0.21)、(5.74±0.29)]及阴性对照组[(7.35±0.62)、(6.02±0.51)]比较,两者显著下降(P<0.05);转染组Bmi-1蛋白表达为(3.31±0.19)较空白对照组的(7.55±0.27)及阴性对照组的(8.10±0.32)下降明显(P<0.05)。结论 Bmi-1siRNA能通过沉默乳腺癌MCF-7细胞Bmi-1基因表达,有效干涉乳腺癌干细胞生物学特性,为乳腺癌干细胞的靶向治疗提供了新思路。
Objective To investigate the effect of B-cell-specific moloney murine leukemia virus insertion site 1 (Bmi-1) gene silencing on the biological characteristics of breast cancer stem cells in MCF-7 breast cancer cells influences. Methods The si RNA expression vector of Bmi-1 gene was constructed and transfected into MCF-7 cells. Stable transfected cells were screened and transfected into cells. The transfected cell lines (negative control group) and blank control group. Serum-free serum-containing suspension culture enriched breast cancer stem cells, preparation of single cell suspension. Western blot was used to detect Bmi-1 protein expression in breast cancer stem cells. The expression of human telomerase reverse transcriptase (hTERT) and vascular endothelial growth factor (VEGF)-C mRNA in each group was detected by RT-PCR. One-way ANOVA analysis of each group were compared, P <0.05 for the difference was statistically significant. Results The expressions of hTERT and VEGF-C mRNA in the transfected group were (3.46 ± 0.50), (2.98 ± 0.18) vs those in the blank control group [(6.88 ± 0.21) and (5.74 ± 0.29) , (6.02 ± 0.51)], respectively. The expression of Bmi-1 protein in the transfected group was significantly higher than that in the blank control group (7.55 ± 0.27 vs. (8.10 ± 0.27) vs ± 0.32) decreased significantly (P <0.05). Conclusion Bmi-1 siRNA can effectively silence the biological characteristics of breast cancer stem cells by silencing the Bmi-1 gene expression in breast cancer MCF-7 cells and provide a new idea for the targeted therapy of breast cancer stem cells.