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目的研究细胞外信号调节激酶-丝裂原激活蛋白激酶途径(ERK-MAPK)与DNA甲基化间的关系及对结肠癌细胞生物学行为的协同影响。方法培养人结肠癌细胞SW1116,分别以PBS、二甲基亚砜(DMSO)为对照组,PD 98059 50μmol／L、5-氮脱氧胞苷(5-aza-dC)5μmol／L、PD 98059 50 μmol／L+5-aza-dC 5μmol／L进行药物干预,以定量RT-PCR检测DNA甲基化酶(Dnmt)1、3a和3b基因转录水平;流式细胞仪分析细胞周期;MTT测定细胞活力;光学显微镜下观察细胞形态学变化。结果 ERK-MAPK途径阻断剂PD 98059下调Dnmt 1和Dnmt 3b,Dnmt抑制剂5 aza-dC下调Dnmt 1、 Dnmt 3a和Dnmt 3b,日5-aza-dC联合PD98059对Dnmt1及Dnmt 3a mRNA的表达下调更为显著。5- aza-dC明显降低G0／G1期细胞百分比(P<0．05),G2／M期细胞百分比明显增加(P<0．05);PD98059 使G0／G1期细胞百分比降低(P<0．05),G2／M期增加(P<0．05)。PD98059明显抑制细胞生长。PD 98059促进细胞分化,呈上皮样改变,细胞变狭长,胞质减少,细胞排列开始出现相对整齐;5-aza-dC干预组细胞大小不一,出现较多多倍体细胞(多个核分裂相)。结论 ERK-MAPK途径阻断剂及Dnmt抑制剂均能抑制结肠癌SW1116细胞分裂、增殖,并诱导细胞分化;两者有协同作,ERK-MAPK信号转导途径能调控DNA甲基化水平。
Objective To investigate the relationship between extracellular signal-regulated kinase-mitogen-activated protein kinase pathway (ERK-MAPK) and DNA methylation and the synergistic effect on colon cancer cell biological behavior. Methods SW1116 human colon cancer cells were cultured with PBS and dimethyl sulfoxide (DMSO) as control groups. PD 98059 50 μmol/L, 5-aza-dC 5 μmol/L, PD 98059 50 5μmol/L μmol/L+5-aza-dC was used for drug intervention to detect the transcription level of DNA methylation (Dnmt) 1, 3a and 3b genes by quantitative RT-PCR; cell cycle was analyzed by flow cytometry; cells were detected by MTT Vitality; observation of cell morphological changes under light microscope. Results PD98059, an ERK-MAPK pathway blocker, downregulated Dnmt 1 and Dnmt 3b, Dnmt 1, Dnmt 3a and Dnmt 3b were down-regulated by Dnmt inhibitor 5 aza-dC, and Dnmt 1 and Dnmt 3a mRNA were expressed by 5-aza-dC and PD98059. The downward adjustment is more pronounced. 5-aza-dC significantly decreased the percentage of cells in G0/G1 phase (P<0.05), the percentage of cells in G2/M phase increased significantly (P<0.05); PD98059 decreased the percentage of cells in G0/G1 phase (P<0). .05) increased in the G2/M phase (P<0.05). PD98059 significantly inhibited cell growth. PD 98059 promoted cell differentiation, showed epithelial-like changes, cells became narrow and long, cytoplasm decreased, and cell alignment began to appear relatively neat; 5-aza-dC intervention group had different cell sizes, and there were many polyploid cells (multiple mitotic phases). . Conclusion Both ERK-MAPK pathway blocker and Dnmt inhibitor can inhibit the cell division and proliferation of colon cancer SW1116 cells and induce cell differentiation. The two have a synergistic effect. ERK-MAPK signal transduction pathway can regulate DNA methylation level.