地西他滨联合丙戊酸钠促胃癌MGC-803细胞nm23-H1基因表达及机制研究

Synergistic Effect of Decitabine and Valproic Acid on Non-metastasis 23-H1 Expression in Gastric MGC-803 Cells

关键词:  nm23-H1基因  MGC-803细胞  丙戊酸钠  地西他滨

Key words:  nm23-H1 gene  MGC-803 cell  decitabine  valproic acid

摘要:

目的 探讨地西他滨(DCA)和丙戊酸钠(VPA)联用对胃癌细胞株MGC-803的作用及对non-metastasis 23-H1基因(nm23-H1)的表达调控的影响。方法 DCA 1.5 及3.0 μmol·L^-1，VPA 1.5 mmol·L^-1，DCA 1.5 μmol·L^-1+VPA 1.5 mmol·L^-1，DCA 3.0 μmol·L^-1+VPA 1.5 mmo·L^-1作用MGC-803细胞72 h。Annexin V/PI法检测细胞凋亡，实时荧光定量PCR检测nm23-H1 mRNA表达，焦磷酸测序法检测nm23-H1启动子上随机选取的两个CpG岛位点甲基化状态。结果 VPA 1.5 +DCA 1.5联合用药组[早期：(33.58±3.88)%，晚期：(31.52±4.20)%]和VPA 1.5 +DCA 3.0联合用药组[早期：(42.61±4.23)%，晚期：(38.01±3.86)%]凋亡率均高于其相应单药组，差异具有统计学意义(P<0.01)。nm23-H1 mRNA在VPA 1.5 +DCA 1.5联合用药组(1.84±0.46)和VPA 1.5 +DCA 3.0联合用药组(2.88±0.42)的表达水平均高于其相应单药组，差异具有统计学意义(P<0.01)。VPA 1.5 +DCA 1.5联合用药组[位点1：(53.50±3.39)%，位点2：(51.17±2.71)%]和VPA 1.5 +DCA 3.0联合用药组[位点1：(41.17±2.14)%，位点2：(39.83±2.56)%]nm23-H1启动子两位点甲基化阳性率均低于其相应单药组，差异具有统计学意义(P<0.01)。VPA 1.5 mmol·L^-1、VPA 1.5+DCA 1.5、VPA 1.5+DCA 3.0这3组的HDAC酶活性均低于正常对照、DCA 1.5 μmol·L^-1、DCA 3.0 μmol·L^-1任一组，差异具有统计学意义(P<0.01)。结论 DCA联合VPA能显著上调nm23-H1基因的表达，其机制与启动子上的甲基化水平降低和去乙酰化酶活性降低有关。

Abstract:

OBJECTIVE To investigate the synergistic effect of decitabine(DCA) and valproic acid(VPA) on non-metastasis 23-H1 (nm23-H1) gene expression in gastric MGC-803 cells. METHODS The groups were set as follows: DCA 1.5 and 3.0 μmol·L^-1, VPA 1.5 mmol·L^-1, DCA 1.5 μmol·L-1+VPA 1.5 mmol·L^-1, DCA 3.0 μmol·L^-1+VPA 1.5 mmo·L^-1. The cells were treated by drug for 72 h. The early and late apoptosis rates were detected by staining with Annexin V and PI. The nm23-H1 mRNA expressions levels were detected by real-time quantity PCR. Methylation status of 2 CpG island selected randomly was detected by pyrosequencing. RESULTS The apoptosis rates of VPA 1.5 +DCA 1.5 group [early: (33.58±3.88)%, late: (31.52±4.20)%] and VPA 1.5+DCA 3.0 [early: (42.61±4.23)%, late: (38.01±3.86)%] were significantly higher than their corresponding concentration single drug groups (P<0.01). The nm23-H1 mRNA relative expression in VPA 1.5+DCA 1.5 group (1.84±0.46) and VPA 1.5+DCA 3.0 group(3.02±0.36) were significantly higher than their corresponding concentration single drug groups(P<0.01). The methylation percentages of 2 CpG islands of nm23-H1 promoter in VPA 1.5 +DCA 1.5 group [site1: (53.50±3.39)%, site2: (51.17±2.71)%] and VPA 1.5+DCA 3.0 group [site1: (41.17±2.14)%, site2: (39.83±2.56)%] were significantly lower than their corresponding concentration single drug groups(P<0.01). The histone deacetylases(HDAC) activity of each of VPA 1.5mmol·L^-1, VPA 1.5+DCA 1.5, VPA 1.5+DCA 3.0 groups was significantly lower than either of Normal control, DCA 1.5 μmol·L^-1, DCA 3.0 μmol·L^-1 groups(P<0.01). CONCLUSION The induction of nm23-H1 mRNA expression by combinations of DCA and VPA is dependent on decreasing the methylation status of nm23-H1 promoter and reducing HDAC activity in gastric MGC-803 cells.