沉默NANOG基因表达对侧群表型肝癌干细胞化疗耐药的影响

目的观察沉默NANOG基因对侧群表型肝癌干细胞化疗耐药的影响，初步探讨提高肝癌化疗效果的新方法。 方法基于侧群（SP）细胞分选法，采用流式细胞术从肝癌细胞Hep3B中分选SP细胞和非侧群（NSP）细胞，通过特异性靶向NANOG基因的siRNA沉默SP细胞中NANOG基因表达，MTT法检测SP细胞对化疗药物阿霉素（DOX）的敏感性，实时荧光定量PCR（real-time PCR）和免疫印迹（Western blotting）检测NANOG和耐药相关基因ABCB1的表达。 结果（1）肝癌细胞Hep3B中分选的SP细胞比例为（13.3±1.8）%。（2）MTT法结果显示DOX处理后SP细胞存活率为（65.3±5.4）%，NSP细胞存活率为（41.2±4.7）%；SP细胞对DOX的耐药性高于NSP细胞（P<0.05）。（3）NANOG和ABCB1 mRNA在SP细胞中的表达水平分别是NSP细胞的4.5、4.0倍；NANOG和ABCB1蛋白在SP细胞中表达水平分别是NSP细胞的4.2、3.6倍，差异均有统计学意义（P<0.05）。（4）RNAi沉默NANOG表达后，siNANOG组SP细胞NANOG、ABCB1的mRNA和蛋白表达水平均较Mock组和siNC组明显下降（P<0.05），SP细胞对阿霉素DOX的耐药性显著降低（P<0.05）。 结论NANOG基因在维持SP表型肝癌干细胞耐药性起重要作用，沉默NANOG表达后SP细胞耐药性受到抑制，可能与ABCB1表达下调有关。

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

ObjectiveTo investigate the effect of NANOG silencing on ABCB1 expression and chemosensitivity of side population (SP) phenotype stem cell-like hepatoma cells to doxorubicin (DOX). MethodsFlow 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. ConclusionsNANOG 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.