Effect of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells

Abstract

Objective 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. Results 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+/CD24-/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. Key words: Breast neoplasms; Transcription factors; Neoplastic stem cells; Suspension culture; RNA interference; Mice