CAJ | 학술논문

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.
목적 이용단발협RNA(shRNA)하조Smoothened(SMO)기인적표체,연구SMO대유선암간세포증식적영향.방법 설계침대인SMO기인적shRNA,전염인유선암MCF-7세포계,G418사선출은정하조SMO기인적세포주.체외:활세포계수시제합(CCK8)검측세포증식적변화,류식세포의검측CD44+/CD24-유선암간세포비례,현부구배양검측유선구형성적능력,Western blot검측SMO、GLI1급Oct4적표체.체내:매3천검측각조라서성류정황,면역조직화학SP법검측SMO적표체,Western blot검측SMO、GLI1급Oct4단백적표체.결과 21 d후사선출은정하조SMO기인적MCF-7세포.CCK8결과현시하조SMO기인가이억제유선암MCF-7세포적증식.류식세포의현시하조SMO기인후,CD44 +/CD24-세포화유선암간세포구형성적비례명현강저.하조후라서적종류생장속도하강.면역조직화학검측현시음성대조조중SMO양성표체비례위5/5,SMO-shRNA조양성비례위2/5.량조종류조직중SMO적단백표체수평분별위0.72 ±0.17화0.21±0.09,GLI1적단백표체수평분별위1.21±0.21화0.47 ±0.12,Oct4적단백표체수평분별위0.83 ±0.13화0.25±0.07.SMO、GLI-1화Oct4재SMO-shRNA조중적표체명현저우음성대조조(P＜0.05).결론 하조SMO기인가이억제유선암간세포적증식.
Objective 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.