Na~+/K~+-ATP酶α1亚单位小干扰RNA和哇巴因诱导肝癌HepG2细胞S期阻滞及其机制

目的 观察哇巴因联合Na~+/K~+-ATP酶(钠泵)α1亚单位小干扰RNA(siRNA)对人肝癌HepG2细胞增殖和细胞周期的影响.方法 分析人肝癌临床组织标本、正常肝组织和肝癌细胞系SMC7721、Bel7402和HepG2细胞钠泵α1亚单位的表达.CCK-8法检测采用钠泵α1亚单位siRNA和哇巴因作用肝癌HepG2细胞增殖抑制状况,分析各组细胞的钠泵活性变化,流式细胞术分析细胞周期变化.实时定量PCR和蛋白质印迹法检测钠泵α1、促分裂源活化蛋白激酶1(MAPK1)、细胞围期蛋白1(CyclinA)、周期蛋白依赖性激酶2(CDK2)、增殖细胞核抗原(PCNA)和细胞周期蛋白抑制因子(P21~(WSF1))表达的变化.结果 肝癌组织标本的钠泵α1亚单位密度值(174.7±16.8)明显高于正常肝组织(65.3±7.9,P<0.05);HepG2细胞钠泵α1亚单位表达明显高SMMC-7721和Bel-7402细胞(P<0.05).siRNA、哇巴因和联合用药可抑制HepG2细胞的增殖,联合实验组效果更显著(P<0.05),0.03μmol/L siRNA组、0.1 μmol/L哇巴因和联合实验组细胞24、48和72 h抑制率分别为17.4%、20.3%、24.3%,37.5%、44.3%、51.2%,52.3%、70.2%、88.2%.各实验组均出现钠泵活性降低,以联合实验组最显著(P<0.05).0.1 μmol/哇巴因和联合实验组HepG2细胞S期比例从24.2%上升至66.5%和75.2%;实时定量PCR和蛋白质印迹检测显示哇巴因组和联合实验组P21<'WAF1>表达上调而钠泵μ1亚单位、MAPK1、CyclinA、CDK2和PCNA表达下调.结论 钠泵α1亚单位siRNA联合哇巴因抑制HepG2细胞钠泵后可抑制细胞增殖,而P21~(WAF1)表达上调和CyclinA/CDK2/PCNA周期调控复合体生成减少可能是引起S期阻滞的主要原因.

Effect of Na~+/K~+-ATPase α1 siRNA and ouabain upon cell cycle in human hepatoma HepG2 cell and its mechanism

Objective To investigate the in vitro anti-cancer effects of ouabin combined Na~+/K~+-ATPase α1 siRNA upon HepG2 cells and its molecular mechanism. Methods The Na~+/K~+ -ATPase α1 subunit expressions of human hepatoma tissue, normal liver tissue and human hepatoma cell lines (HepG2, SMC7721 and Bel7402) were determined. The cells received different treatments (0. 03 μmol/L siRNA, 0. 1 μmol/L ouabain and combination). The proliferation of HepG2 was observed by CCK-8 assay. The activity of Na~+/K~+-ATPase was measured. The HepG2 cell cycle distribution was detected by flow cytometry. The mRNA and protein levels of Na~+/K~+-ATPase α1 subunit, MAPK1, CyclinA, CDK2, PCNA and P21WAF1 were detected by quantitative RT-PCR and Western blot. Results The gray value Na~+/K~+ -ATPase α1 subunit in hepatoma tissue was 174. 74 ± 16. 77 and 65.31 ± 7. 88 respectively in normal liver tissue. The Na~+/K~+ -ATPase α1 subunit expression of hepatoma tissue was significantly higherthan normal liver tissue(P < 0. 05). The Na~+/K~+ -ATPase α1 subunit expression level of HepG2 was higher than that in SMMC-7721 and Bel-7402 cells. The CCK-8 experiments demonstrated that siRNA, ouabain and combination could inhibit the HepG2 proliferation, and the combination group was different from the ouabain or siRNA group (P < 0. 05). The 24, 48 and 72 h inhibitory rates of 0. 03 μmol/L siRNA, 0. 1 μmol/L ouabain and combination group were (17.4%, 20. 3%, 24. 3%), (37. 5%, 44. 3%, 51.2%) and (52. 3% ,70. 2% ,88. 2%) respectively. The activity of Na~+/K~+ -ATPase decreased in siRNA, ouabain and combination group. The S phase proportion of ouabain and combination group increased from 24. 2% to 66. 5% and 75. 2% respectively. The 0. 1 μmol/L ouabain and combination group could down-regulate the expression of Na~+/K~+-ATPsae α1, MAPK1, CyclinA, CDK2, PCNA and up-regulate the expression of P21WAF1 in HepG2 cell. Conclusion The Na~+/K~+ -ATPase α1 siRNA combined ouabain inhibits the proliferation of HepG2 cells by decreasing the expression of MAPK1 and induces the cell cycle S arrest by decreasing the production of CyclinA/CDK2/PCNA complex and increasing the expression of P21~(WAF1).