Abstract: BACKGROUND: Previous studies have shown that peroxisome proliferator-activated receptor gamma coactivator 1 beta (PGC-1β) affected the proliferation, migration and apoptosis of breast cancer cells through PI3K/AKT/mTOR signaling pathway. However, whether PGC-1β can affect the stemness of breast cancer stem cells through PI3K/AKT/mTOR signaling pathway and its influencing mechanism has not been elucidated.
OBJECTIVE: To investigate the effect and regulatory mechanism of PGC-1β on stemness of breast cancer stem cells.
METHODS:  MCF-7 cells were transfected with PGC-1β empty vector, PGC-1β overexpression vector (Lv-PGC-1β) and interference vector (Sh-PGC-1β), respectively. The transfection effect was observed under fluorescence microscope. The relative expression of PGC-1β mRNA and protein after lentivirus transfection was verified by qRT-PCR and western blot assay. The cells of non-transfection group, PGC-1β empty vector group, PGC-1β overexpression vector group and PGC-1β interference vector group were cultured with stemness medium to foster breast cancer stem cells. The formation process of the spheres was observed and recorded under the microscope. Western blot assay was used to verify the relative expression of stemness proteins (ABCG2, ALDH1 and OCT-4), epithelial-mesenchymal transition proteins (E-cadherin, N-cadherin, Vimentin, Slug and ZEB1) and PI3K/AKT/mTOR pathway related proteins.
RESULTS AND CONCLUSION: (1) MCF-7 adherent cells were cultured in stemness medium to form dense sphere stem cells with good refractive index and high intermediate density. (2) The expression of stemness protein in breast cancer stem cells was significantly higher than that in adherent cells (P < 0.01). (3) Compared with the non-transfection and PGC-1β empty vector groups, the formation time of breast cancer stem cells was shorter, and the number and diameter of spheres were increased in the PGC-1β overexpression vector group (P < 0.01); however, the number of spheroids and the diameter of spheroids decreased in the PGC-1β interference vector group (P < 0.01). (4) The expression of stemness-related proteins in PGC-1β overexpression group was higher than that in non-transfection group and PGC-1β empty vector group (P < 0.01). Epithelial-mesenchymal transition protein E-Cadherin expression was lower in the PGC-1β overexpression vector group than that in the non-transfection group and PGC-1β empty vector group (P < 0.01). The expression levels of N-Cadherin, Vimentin, Slug and ZEB1 were higher in the PGC-1β overexpression vector group than those in the non-transfection group and the PGC-1β empty vector group (P < 0.01). The expression levels of PI3K/AKT/mTOR-related proteins and their phosphorylation-related proteins were higher in the PGC-1β overexpression vector group than those in the non-transfection group and PGC-1β empty vector group (P < 0.01). The results of the PGC-1β interference vector group were opposite to that of the PGC-1β overexpression vector group. (5) These results suggest that PGC-1β can promote the expression of stemness genes and proteins in breast cancer stem cells by activating the PI3K/AKT/mTOR signaling pathway and affecting epithelial-mesenchymal transition.

Key words: PGC-1β, MCF-7 cell, breast cancer stem cell, stemness, transfection, epithelial-mesenchymal transition, PI3K/AKT/mTOR signaling pathway, mammosphere experiment