Sorting And Identifying The Group Of Cancer Stem Cells In Cell Line ACC-M

Objective Observing EMT-related and CSCs-related biomarkers by Q-PCR before and afer sorting by FCM in cell line ACC-M, which was marked with ABCG2, we primarily sorted and discussed the group of cancer stem cells(CSCs) in cell line ACC-M combining the theories of epithelial-mesenchymal transition(EMT) and CSCs.Methods (1) Q-PCR dynamic observations of the EMT-related biomarkers E-cadherin, N-cadherin, Vimentin, MMP-2and CSCs-related biomarkers Bmi-1, Oct-4, CD44, CD24on ACC-M cells during72hours of induction and culture of lOng/ml TGF-β1.(2) Flow cytometry(FCM) technique was employed to examine the regulation of ABCG2+subpopulation by TGF-β1.(3)6groups of cells were sorted by FCM: A1:TGF-β1induced、B1:TGF-β1induced+、A2:TGF-β1induced-ABCG2+、 B2:TGF-β1induced+ABCG2+、A3:TGF-β1induced-ABCG2-、B3:TGF-β1induced+ABCG2-, and their EMT-related and CSCs-related biomarkers were examined by Q-PCR.Results (1) EMT-related biomarkers:E-cadherin, a biomarker of epithelial phenotype, gradually decreased with the induction time while N-cadherin and Vimentin, biomarkers of mesenchymal phenotype, and MMP-2, an EMT cofactor, all gradually increased with the induction time; CSCs-related biomarkers, Oct-4, a transfactor, and CD24, a adhesion molecule, gradually decreased with the induction time while Bmi-1, a proto-oncogene, and CD44, a adhesion molecule, gradually increased with the induction time; the onset of EMT was closely correlated with the expression of CSCs-related biomarkers.(2)After the induction and culture of TGF-β1, the proportion of ABCG2+cells significantly increased from3.06%to6.66%.(3) Longitudinal comparison:①The expression of E-cadherin, before and after the induction of TGF-β1, was highest in ABCG2-group, lower in non-sorting group, lowest in ABCG2+group;②The expressions of N-cadherin, MMP-2, Bmi-1and Oct-4, before and after the induction, were lowest in ABCG2-group, higher in non-sorting group, highest in ABCG2+group;③The expression of CD44were not statistically variant among groups before TGF-(31induction, but after the induction, its expression was highest in ABCG2+group. There is no statistical variance between ABCG2-group and non-sorting group.④The variant expressions of Vimentin and CD24were not statistically significant among all3groups before or after the induction. Lateral comparison:①TGF-β1induced+groups had comparatively lower expressions of E-cadherin, Oct-4and CD24than TGF-β1induced-groups;②TGF-β1induced+groups had comparatively higher expressions of N-cadherin, Vimentin, MMP-2, Bmi-1and CD44than TGF-β1induced-groups. Conclusions (1) TGF-β1can induce ACC-M cells to undergo EMT. Meanwhile, the phenotypic expression of SP cells in ACC-M cells is enhanced, and SP cells are more prone to EMT than non-SP cells.(2) Bmi-1, Oct-4, CD44, CD24participate in the regulation of the proportion of CSCs cells and Bmi-1might be a specific biomarker of CSCs in ACC-M cells.