Mao Jun, Fan Pan-hong, Ma Wei, Zhang Qing-qing, Wang Bo, Fan Shu-jun, Li Lian-hong
The Center of Clinical Pathology, Dalian Medical University, Dalian 116044, China.
Zhonghua Bing Li Xue Za Zhi. 2013 Apr;42(4):262-6. doi: 10.3760/cma.j.issn.0529-5807.2013.04.011.
To investigate the influence of down-regulating Smoothened (SMO) gene expression through short hairpin RNA (shRNA) on the proliferation of breast cancer stem cells.
Human SMO shRNA was designed, synthesized chemically, and transfected into MCF-7 cells to down-regulate SMO gene. By using G418, stable cells with down-regulated SMO were selected. In vitro proliferation of these cells was measured by CCK8 assay. The proportion of CD44(+)/CD24(-) cells was detected by flow cytometry and the mammospheres formation was determined by suspension sphere culture. The expression of SMO, GLI1 and Oct4 was detected by Western blot. In vivo, the volume of tumor was measured every 3 days and the expression of SMO, GLI1 and Oct4 detected by Western blot.
In vitro, the cells were transfected with SMO-shRNA and selected by G418 after 21 days. SMO-shRNA effectively down-regulated the expression of SMO gene and protein, and inhibited the proliferation of MCF-7 and markedly reduced the proportion of CD44(+)/CD24(-) cells and mammospheres. In vivo, SMO-shRNA treatment of MCF-7 significantly inhibited the volume of tumor. The positive rate of SMO in negative control and SMO-shRNA group was 5/5 and 2/5, respectively. The expression of SMO, GLI1 and Oct4 in different groups were 0.72 ± 0.17 and 0.21 ± 0.09, 1.21 ± 0.21 and 0.47 ± 0.12, 0.83 ± 0.13 and 0.25 ± 0.07. SMO, GLI1 and Oct4 down-regulation significantly suppressed at protein levels (P < 0.05).
The shRNA by chemical synthesis can effectively down-regulate SMO gene expression and inhibit the proliferation of breast cancer stem cells.
研究通过短发夹RNA（shRNA）下调平滑肌瘤（SMO）基因表达对乳腺癌干细胞增殖的影响。
设计人SMO shRNA，化学合成后转染至MCF-7细胞以下调SMO基因。利用G418筛选出SMO下调的稳定细胞。采用CCK8法检测这些细胞的体外增殖情况。通过流式细胞术检测CD44(+)/CD24(-)细胞比例，采用悬浮球培养法测定成球能力。通过蛋白质免疫印迹法检测SMO、GLI1和Oct4的表达。在体内，每3天测量一次肿瘤体积，并通过蛋白质免疫印迹法检测SMO、GLI1和Oct4的表达。
体外实验中，细胞转染SMO-shRNA后21天用G418筛选。SMO-shRNA有效下调SMO基因和蛋白表达，抑制MCF-7细胞增殖，并显著降低CD44(+)/CD24(-)细胞比例和成球能力。在体内，SMO-shRNA处理MCF-7细胞显著抑制肿瘤体积。阴性对照组和SMO-shRNA组中SMO的阳性率分别为5/5和2/5。不同组中SMO、GLI1和Oct4的表达分别为0.72±0.17和0.21±0.09、1.21±0.21和0.47±0.12、0.83±0.13和0.25±0.07。SMO、GLI1和Oct4的下调在蛋白水平上显著受到抑制（P<0.05）。
化学合成的shRNA可有效下调SMO基因表达并抑制乳腺癌干细胞的增殖。
