Silencing of NANOG expression enhances doxorubicin sensitivity in the side population phenotype stem cell-like hepatoma cells

Abstract

Objective To investigate the effect of NANOG silencing on ABCB1 expression and chemosensitivity of side population (SP) phenotype stem cell-like hepatoma cells to doxorubicin (DOX). Methods Flow cytometry was used to sort SP cells and non-side population (NSP) cells from hepatoma cell line Hep3B. Transient transfection of specific siRNA targeting NANOG gene into SP cells was performed, and chemosensitivity of SP cells and NSP cells to DOX were tested by MTT assay, expression levels of NANOG and ABCB1 mRNA and protein in SP cells and NSP cells were detected by Real-time PCR and Western blotting. Results (1) The percentage of SP cells in the hepatoma cell line Hep3B was (13.3±1.8)%. (2) MTT assay showed that SP cells had stronger chemoresistance to DOX than NSP cells [(65.3±5.4)% vs (41.2±4.7)%, P<0.05]. (3) The expression levels of NANOG and ABCB1 mRNA in SP cells were 4.5 and 4.0 times higher than that in NSP cells. The expression levels of NANOG and ABCB1 proteins in SP cells were 4.2 and 3.6 times higher than that in NSP cells, respectively (P<0.05). (4) Transfection of NANOG siRNA into SP cells effectively inhibited the expression of NANOG mRNA and protein. Compared with Mock and siNC group, the siRNA-mediated silencing of NANOG expression in SP cells resulted in decreased chemoresistance to DOX (P<0.05). Furthermore, knockdown of NANOG led to the down-regulation of ABCB1 mRNA and protein expression. Conclusions NANOG plays a role in chemoresistance of SP phenotype stem cell-like hepatoma cells. Silencing of NANOG expression enhanced DOX sensitivity in the SP phenotype stem cell-like hepatoma cells, at least in part through downregulating ABCB1 expression. Key words: Liver neoplasms; Neoplastic stem cells; NANOG; ABCB1; Drug resistance, neoplasm