Objce tive To investigate the effect and significance of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells.Methods Breast cancer cell line MDA-MB-231 cells were used in this study.Breast cancer stem cells were isolated and enriched by serum-free culture.The obtained stem cells were identified through calculating the percentages of CD44 and CD24 stem cells by FACS and evaluating the paclitaxel resistance in vitro and tumorigenicity in mice.RT-PCR, real-time PCR (qPCR) and Western blot were used to detect Oct4 expression.RNA interference was applied to induce Oct4 down-regulation.The interference experiment set up a control group ( no siRNA transfection) , negative control group ( negative siRNA group,transfection of siRNA sequences without any interfering effect on the cells) and Oct4 siRNA group ( transfection of siRNA with interfering effect on the Oct4 gene) .Methyl thiazolyl tetrazolium ( MTT ) and Transwell chamber tests were conducted to detect the proliferation and invasion ability of MDA-MB-231 breast cancer stem cells after Oct4 knock-down, and paclitaxel inhibition test was applied to evaluate drug resistance of MDA-MB-231 breast cancer stem cells after Oct4 knock-down. Resulst MDA-MB-231 breast cancer stem cells grew as spheres cultured in serum-free suspension.MDA-MB-231 breast cancer stem cells showed a higher percentage of CD44+C/D24 -/low cells (97.2%) than that in MDA-MB-231 breast cancer cells ( 76.6%) ( P<0.05) .The tumor size in mice inoculated with MDA-MB-231 breast cancer stem cells was (124.60±13.65)mm3, significantly larger than that of mice inoculated with breast cancer cells (68.20±9.99 mm3) (P=0.0007).MDA-MB-231 breast cancer stem cells were less sensitive to paclitaxel inhibition than MDA-MB-231 breast cancer cells showing by 50% inhibitory concentration (IC50) [(4.40±0.48) μg/ml vs.(8.20±0.34) μg/ml, P<0.05].However, the expression of transcriptional factors Oct4 was higher in MDA-MB-231 breast cancer stem cells than that in breast cancer cells (P<0.05).The proliferation potential of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was significantly lower than that in the negative siRNA and control groups ( P<0.05) from the third day.The invasion ability of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was obviously reduced than that in the control and negative siRNA groups shown by number of penetrated cells [(46.52±2.58) vs.(79.67±3.85) and (77.29±2.13), P<0.05 for both].As for resistance to paclitaxel, IC50 of MDA-MB-231 breast cancer stem cells with Oct4siRNA interference was significantly decreased [(4.48±0.22) μg/ml] compared with that in the control [(7.99±0.59) μg/ml] and negative siRNA group [(8.10±0.68) μg/ml] (P<0.05 for both).Conclusions MDA-MB-231 breast cancer cells are successfully obtained by serum-free culture. The proliferation potential, invasion ability and drug resistance of breast cancer stem cells were down-regulated by Oct4 gene knock-down.

Objce tive To investigate the effect and significance of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells.Methods Breast cancer cell line MDA-MB-231 cells were used in this study.Breast cancer stem cells were isolated and enriched by serum-free culture.The obtained stem cells were identified through calculating the percentages of CD44 and CD24 stem cells by FACS and evaluating the paclitaxel resistance in vitro and tumorigenicity in mice.RT-PCR, real-time PCR (qPCR) and Western blot were used to detect Oct4 expression.RNA interference was applied to induce Oct4 down-regulation.The interference experiment set up a control group ( no siRNA transfection) , negative control group ( negative siRNA group,transfection of siRNA sequences without any interfering effect on the cells) and Oct4 siRNA group ( transfection of siRNA with interfering effect on the Oct4 gene) .Methyl thiazolyl tetrazolium ( MTT ) and Transwell chamber tests were conducted to detect the proliferation and invasion ability of MDA-MB-231 breast cancer stem cells after Oct4 knock-down, and paclitaxel inhibition test was applied to evaluate drug resistance of MDA-MB-231 breast cancer stem cells after Oct4 knock-down. Resulst MDA-MB-231 breast cancer stem cells grew as spheres cultured in serum-free suspension.MDA-MB-231 breast cancer stem cells showed a higher percentage of CD44+C/D24 -/low cells (97.2%) than that in MDA-MB-231 breast cancer cells ( 76.6%) ( P<0.05) .The tumor size in mice inoculated with MDA-MB-231 breast cancer stem cells was (124.60±13.65)mm3, significantly larger than that of mice inoculated with breast cancer cells (68.20±9.99 mm3) (P=0.0007).MDA-MB-231 breast cancer stem cells were less sensitive to paclitaxel inhibition than MDA-MB-231 breast cancer cells showing by 50% inhibitory concentration (IC50) [(4.40±0.48) μg/ml vs.(8.20±0.34) μg/ml, P<0.05].However, the expression of transcriptional factors Oct4 was higher in MDA-MB-231 breast cancer stem cells than that in breast cancer cells (P<0.05).The proliferation potential of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was significantly lower than that in the negative siRNA and control groups ( P<0.05) from the third day.The invasion ability of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was obviously reduced than that in the control and negative siRNA groups shown by number of penetrated cells [(46.52±2.58) vs.(79.67±3.85) and (77.29±2.13), P<0.05 for both].As for resistance to paclitaxel, IC50 of MDA-MB-231 breast cancer stem cells with Oct4siRNA interference was significantly decreased [(4.48±0.22) μg/ml] compared with that in the control [(7.99±0.59) μg/ml] and negative siRNA group [(8.10±0.68) μg/ml] (P<0.05 for both).Conclusions MDA-MB-231 breast cancer cells are successfully obtained by serum-free culture. The proliferation potential, invasion ability and drug resistance of breast cancer stem cells were down-regulated by Oct4 gene knock-down.