RNAi抑制NANOG表达对肝癌细胞HepG2中MDR1表达及阿霉素敏感性的影响

RNAi knockdown of NANOG inhibits MDR1 expression and promotes doxorubicin sensitivity in human hepatoma HepG2 cells

目的探讨抑制NANOG表达对肝癌细胞HepG2中多药耐药基因1(multidrug resistance gene 1,MDR1)表达及阿霉素敏感性的影响。方法将靶向NANOG基因的特异性siRNA转染肝癌细胞HepG2,Real-time PCR和Western blot检测siRNA沉默NANOG基因后NANOG、MDR1 mRNA和蛋白的表达,平板克隆形成实验检测沉默NANOG基因后对细胞增殖能力的影响,流式细胞术检测沉默NANOG基因后对细胞周期的影响,CCK-8法检测沉默NANOG基因后HepG2细胞对阿霉素的敏感性情况。结果靶向NANOG基因特异性siRNA转染肝癌细胞HepG2后,能有效抑制NANOG mRNA和蛋白表达,与Mock组比值分别为(0.32±0.05)和(0.38±0.08);与Mock组比较,沉默NANOG基因后,细胞克隆形成率下降[(8.51±3.63)%vs(17.13±2.24)%,P<0.05],进入G0/G1期的细胞比例增多[(75.33±8.21)%vs(57.81±5.05)%,P<0.05],HepG2细胞对阿霉素的敏感性增强(P<0.05),HepG2细胞内MDR1 mRNA和蛋白表达下降,与Mock组比值分别为(0.35±0.06)和(0.41±0.08)。结论抑制NANOG表达可引起肝癌细胞HepG2中MDR1的表达下调并增强细胞对阿霉素的敏感性。

Objective To investigate the effect of NANOG knockdown on multidrug resistance gene 1 （MDR1） expression and doxorubicin chemosensitivity of human hepatocarcinoma HepG2 cells.Methods Transient transfection of the specific siRNA targeting NANOG gene into HepG2 cells was performed. The expression of NANOG and MDR1 at mRNA and protein levels was detected by real-time PCR and Western blotting. The influence of NANOG knockdown on cell proliferation ability was examined by colony formation assay. The influence on cell cycle was tested by flow cytometry. The doxorubicin chemosensitivity of HepG2 cells was detected by CCK-8 assay.Results Transfection of the specific siRNA targeting NANOG gene into HepG2 cells could effectively inhibit the expression of NANOG mRNA and protein, with the ratio of siRNA group to mock group of 0.32±0.05 and 0.38±0.08, respectively. Compared with mock group, the siRNA-mediated silencing of NANOG gene in HepG2 cells resulted in decreased cell clone formation rate [（8.51±3.63）% vs （17.13±2.24）%, P〈0.05], increased proportion of cells in G0/G1 phase [（75.33±8.21）% vs （57.81±5.05）%, P〈0.05], and increased chemosensitivity to doxorubicin （P〈0.05）. Furthermore, knockdown of NANOG gene caused the down-regulation of MDR1 mRNA and protein expression, with the ratio of siRNA group to mock group of 0.35±0.06 and 0.41±0.08, respectively.Conclusion Inhibition of NANOG expression in HepG2 cells causes down-regulation of MDR1 expression and enhances the chemosensitivity of cells to doxorubicin.