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目的探讨DNA5’CpG岛去甲基化对RKO结肠癌细胞株maspin基因的转录调控作用及对细胞株生长增殖的生物学影响,寻找抗癌治疗的新靶点。方法应用甲基化特异性PCR(methylationspecificPCR,MSP)检测RKO结肠癌细胞株maspin基因核心启动子区CpG岛甲基化情况;用特异性DNA甲基转移酶抑制剂5-氮-2’-脱氧胞苷(5-aza-2’-deoxycytidine,5-aza-CdR)作用结肠癌细胞株72h后,逆转录聚合酶链反应(RT-PCR)检测maspinmRNA表达,四唑盐(MTT)比色、流式细胞仪检测用药前后RKO细胞株生长存活率和细胞周期的变化。结果RKO结肠癌细胞株maspin基因核心启动子区存在CpG岛甲基化。与对照组相比,3组不同浓度5-aza-CdR作用后,maspin基因mRNA表达分别增加了10.89、16.91和23.97倍,明显抑制肿瘤细胞生长,阻滞细胞周期于G0/G1期,诱导细胞凋亡,凋亡率分别为5.17%、8.71%和11.23%。结论RKO结肠癌细胞株maspin基因5’端核心启动子甲基化可能是导致该基因表达沉默的主要原因;特异性甲基转移酶抑制剂5-aza-CdR能较好地逆转RKO细胞DNA异常甲基化,并有效地激活因高甲基化所致maspin基因沉默的再转录,诱导该基因表达,从而抑制肿瘤细胞生长,诱导细胞凋亡。
OBJECTIVE: To investigate the transcriptional regulation of maspin gene in RKO colon cancer cell line and its biological effects on the growth and proliferation of RKO colon cancer cell line by demethylation of DNA 5’CpG island, and to search for a new target of anti-cancer therapy. Methods Methylation specific PCR (MSP) was used to detect CpG island methylation in the core promoter region of maspin gene in RKO colon cancer cell line. Methylation of 5-Aza-2’-deoxy The colon cancer cell lines were treated with 5-aza-2-deoxycytidine (5-aza-CdR) for 72 hours, and maspin mRNA expression, MTT colorimetric assay, Flow cytometry assay before and after treatment RKO cell growth survival rate and cell cycle changes. Results CpG island methylation was found in the core promoter region of maspin gene in RKO colon cancer cell line. Compared with the control group, the mRNA expression of maspin gene increased by 10.89, 16.91 and 23.97 folds after treated with 5-aza-CdR at different concentrations for 3 different concentrations, which significantly inhibited the growth of tumor cells, blocked the cell cycle at G0 / G1 phase, induced cells The apoptotic rates were 5.17%, 8.71% and 11.23% respectively. Conclusion Methylation of the 5 ’core promoter of RCC colon cancer cell line may be the main reason for the silencing of this gene. Specific methyltransferase inhibitor 5-aza-CdR can reverse DNA abnormality in RKO cells Methylation and effectively activate the transcription of maspin gene silencing induced by hypermethylation to induce the gene expression, thereby inhibiting tumor cell growth and inducing apoptosis.