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目的检测不同分化程度胃痛细胞株runt相关转录因子3(RUNX3)基因及其甲基化表达.观察RUNX3 mRNA再表达对胃癌细胞株生物学特性的影响。方法5-氮杂脱氧胞苷(5-AZA-dC)化学处理法和甲基化敏感性聚合酶链反砬(MSP法)检测胃癌细胞RUNX3基因的甲基化状态;采用四甲基偶氮唑盐(MTT)实验检测5氟尿嘧啶(5-FU)对5-AZA-dC处理前、后细胞株的生长抑制率,流式细胞术检测转化生长因子β1(TGF-β1)诱导凋亡的作用。结果①RUNX3在人胃癌细胞SGC7901、MKN45、BGC823中表达.而在MKN28中表达沉默,DNA异常甲基化仪存在于MKN28细胞;②5-AZA-dC处理后,MKN28细胞生长速度显著慢于未处理组,不同浓度5-FU对5-AZA-dC处理后MKN28的生长抑制率显著高于未处理组(P<0.05);③TGF-β1诱导5 AZA-dC处理前后MKN28细胞的早期凋亡具有时效性(P<0.05).两者联用具有协同凋亡作用(P<0.05)。结论DNA异常甲基化是MKN28细胞RUNX3表达沉默的重要机制;RUNX3甲基化可被去甲基化剂5-AZA-dC有效逆转;RUNX3 mRNA的再表达可增强MKN28细胞对5-FU的敏感性和对TGF-β1诱导细胞凋亡的能力。
Objective To detect the gene and its methylation of runt-associated transcription factor 3 (RUNX3) gene in gastric cancer cell lines with different degrees of differentiation.Observe the effect of RUNX3 mRNA expression on the biological characteristics of gastric cancer cell lines. Methods The methylation status of RUNX3 gene in gastric cancer cells was detected by 5-AZA-dC chemiluminescence assay and methylation-sensitive polymerase chain reaction (MSP method) The growth inhibition rate of 5-AZA-dC treated 5-AZA-dC treated with 5-fluorouracil (5-FU) was measured by MTT assay and the apoptosis induced by TGF-β1 was detected by flow cytometry . RESULTS: ①RUNX3 was expressed in human gastric cancer cell lines SGC7901, MKN45 and BGC823, while silenced in MKN28 and abnormal DNA methylation in MKN28 cells. ② After 5-AZA-dC treatment, MKN28 cells grew more slowly than untreated cells The inhibitory effect of 5-AZA-dC with different concentrations of 5-AZA on the growth of MKN28 cells was significantly higher than that of untreated cells (P <0.05). ③The early apoptosis of MKN28 cells induced by 5-AZA-dC induced by TGF- (P <0.05) .The combination of the two had synergistic apoptosis (P <0.05). Conclusions DNA abnormal methylation is an important mechanism of RUNX3 silencing in MKN28 cells. RUNX3 methylation can be effectively reversed by demethylating agent 5-AZA-dC; re-expression of RUNX3 mRNA can enhance the sensitivity of MKN28 cells to 5-FU And on TGF-β1-induced apoptosis.