摘要目的 利用短发夹RNA(shRNA)下调Smoothened(SMO)基因的表达,研究SMO对乳腺癌干细胞增殖的影响.方法 设计针对人SMO基因的shRNA,转染人乳腺癌MCF-7细胞系,G418筛选出稳定下调SMO基因的细胞株.体外:活细胞计数试剂盒(CCK8)检测细胞增殖的变化,流式细胞仪检测CD44+/CD24-乳腺癌干细胞比例,悬浮球培养检测乳腺球形成的能力,Western blot检测SMO、GLI1及Oct4的表达.体内:每3天检测各组裸鼠成瘤情况,免疫组织化学SP法检测SMO的表达,Western blot检测SMO、GLI1及Oct4蛋白的表达.结果 21 d后筛选出稳定下调SMO基因的MCF-7细胞.CCK8结果显示下调SMO基因可以抑制乳腺癌MCF-7细胞的增殖.流式细胞仪显示下调SMO基因后,CD44 +/CD24-细胞和乳腺癌干细胞球形成的比例明显降低.下调后裸鼠的肿瘤生长速度下降.免疫组织化学检测显示阴性对照组中SMO阳性表达比例为5/5,SMO-shRNA组阳性比例为2/5.两组肿瘤组织中SMO的蛋白表达水平分别为0.72 ±0.17和0.21±0.09,GLI1的蛋白表达水平分别为1.21±0.21和0.47 ±0.12,Oct4的蛋白表达水平分别为0.83 ±0.13和0.25±0.07.SMO、GLI-1和Oct4在SMO-shRNA组中的表达明显低于阴性对照组(P＜0.05).结论 下调SMO基因可以抑制乳腺癌干细胞的增殖.

abstractsObjective To investigate the influence of down-regulating Smoothened (SMO) gene expression through short hairpin RNA (shRNA) on the proliferation of breast cancer stem cells.Methods Human SMO shRNA was designed,synthesized chemically,and transfected into MCF-7 cells to downregulate 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.Results 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).Conclusion The shRNA by chemical synthesis can effectively down-regulate SMO gene expression and inhibit the proliferation of breast cancer stem cells.