Silencing Twist gene leads to mesenchymal-to-epithelial transition and loss of stemlike properties in breast cancer

Abstract

Objective To observe the effect of silencing Twist gene on mesenchymal-to-epithelial transition (MET) and stem-like properties in breast cancer. Methods The small interfering RNA (siRNA) vectors targeting Twist gene were transfected into MDA-MB-231 cells. The mRNA and protein expression levels of Twist and MET-associated genes (Slug, Snail and Vimentin) were detected by using real-time quantitative polymerase chain reaction(Real-time PCR) and Western blotting. Cell morphology was observed under microscope. The proportion of CD44+ CD24- cells was measured by flow cytometry. In vitro self-renewal capacity was evaluated by sphere formation assay. Tumor-initiating ability was measured by limited dilution assays. Results Compared to the control group, the Twist mRNA (0.24±0.04, P<0.01) and protein expression in siRNA group was significantly decreased. Cells with Twist silencing underwent MET reversal, showing an epithelial-like appearance, decreased mRNA expression of mesenchymal markers, including Slug (0.25±0.05, P<0.01), Snail (0.34±0.03, P<0.01) and Vimentin (0.44±0.04, P<0.01), increased mRNA expression of epithelial marker, E-cadherin (58.71±1.27, P<0.01), down-regulated protein levels of Slug, Snail and Vimentin, and up-regulated protein level of E-cadherin. The proportion of CD44+ CD24- cells in the si-Twist group and control group was (63.30±0.80)% and (86.50±0.70)% respectively (P<0.01). The sphere number in the si-Twist and control groups was (46.67±2.52) and (109.67±2.52) respectively (P<0.01). Twist silencing led to the decrease of about 6-folds in tumor-initiating ability in nude mice compared to the control (P<0.01). Conclusion Silencing Twist gene leads to MET and loss of self-renewal capacity and tumor-initiating ability in breast cancer. Key words: Breast cancer; Twist; Small interfering RNA; Mesenchymal-to-epithelial transition; Tumor stem cells