Epithelial-mesenchymal transition is associated with increased invasiveness of side population cells from hepatoma SMMC-7721 cells

Abstract

To study the in vitro invasiveness of side population (SP), cells purified from hepatoma SMMC-7721 cells and the expressions of epithelial-mesenchymal transition (EMT) markers of SP cells and non-SP (NSP) cells, fluorescence-activated cell sorting (FACS) was used to sort SP cells and NSP cells from SMMC-7721 cells. The in vitro invasiveness of both subpopulations was tested by Transwell chamber assay. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot were applied to evaluate the expressions of Vimentin, E-cadherin, N-cadherin and Snail in SP cells and NSP cells, respectively. In Transwell invasion assay, the number of SP cells (66.3 ± 7.4) penetrating the basilar membrane was significantly higher than that of NSP cells (27.5 ± 6.5) (P < 0.01). The mRNA expressions of Vimentin, N-cadherin and Snail was significantly increased in SP cells (9.527-fold, 6.834- fold and 8.173-fold, respectively) when compared with that in NSP cells (P < 0.01). In contrast, the decrease of E-cadherin mRNA expression in SP cells (5.353-fold) was markedly higher than that in NSP cells (P<0.01). The protein expressions of E-cadherin, Vimentin, N-cadherin and Snail in SP cells were markedly different from those in NSP cells (0.174 ± 0.014 and 0.935 ± 0.012; 1.117 ± 0.012 and 0.314±0.011; 0.975 ± 0.017 and 0.179 ± 0.013; 0.917 ± 0.014 and 0.202 ± 0.013; P < 0.01). The results suggest that SP cells from hepatoma SMMC-7721 cells are highly invasive in vitro which may be related to the changes in expressions of EMT markers.

Key words: SMMC-7721, cancer stem cells, side population cells, invasion, epithelial-mesenchymal transition.