地西他滨调控舌癌细胞RUNX3基因甲基化

Effects of 5-aza-2-deoxycytidine on Methylation Status of RUNX3 Gene in Tongue Cancer SCC-4 Cells

目的:探讨经典甲基化酶抑制剂地西他滨(5-氮杂-2′-脱氧胞苷5-aza-2-deoxycytidine,5-AzaCdR)对舌鳞状细胞癌细胞系SCC-4中抑癌基因RUNX3基因甲基化水平的影响。方法:用不同浓度的地西他滨处理舌癌细胞株SCC-4,72h后甲基化特异性PCR(MSP)检测SCC-4细胞RUNX3基因的甲基化状态,实时定量qPCR技术检测SCC-4细胞RUNX3基因mRNA的表达,Western blot检测RUNX3、p53及裂解PARP蛋白的表达。结果:MSP检测未处理组细胞RUNX3基因呈高甲基化状态,经地西他滨处理后甲基化得到逆转;qPCR检测显示不同浓度地西他滨处理SCC-4细胞72h后相对mRNA表达随着药物浓度增加而增加(P<0.05);Western blot分析结果显示不同浓度的地西他滨组的RUNX3蛋白表达相对水平随药物浓度增加而增加,并且增加p53及裂解PARP蛋白水平。结论:地西他滨可逆转舌癌SCC-4细胞中RUNX3基因的高甲基化状态,并恢复RUNX3基因mRNA及蛋白的表达,促进细胞凋亡。

Objective： To investigate the effects of 5-aza-2-deoxycytidine （5-Aza-CdR） on methylation sta- tus and expression of RUNX3 gene in tongue cancer SCC-4 cells. Methods： Tongue cancer cell line SCC-4 cells were treated with 5-Aza-CdR for different concentrations. After 5-Aza--CdR treatment for 72 h methylation status of RUNX3 gene of SCC-4 cells were detected by methylation specific PCR （MSP）, the expression of RUNX3 gene mRNA were detected by real-time quantitative PCR, the expression of RUNX3, p53 and cleaved PARP protein were detected by Western blot. Results： The results showed that RUNX3 gene of SCC--4 cells was in high methylation status in untreated group, abnormal methylation was effectively reversed by 5-Aza--CdR treat- ment in a dose-dependent manner. After different concentration of 5-Aza-CdR treatment for 72 h, relative mR- NA expression level were increased gradually （P〈O. 05）. The relative expression levels of RUNX3 protein in 5- Aza-CdR treated group were significantly higher as compared with control group. Western blot confirmed in- creased p53 and cleaved PARP protein level in 5-Aza-CdR treated group in a dose dependent manner. Conclusion： The data suggested that 5-Aza-CdR treatment for tongue cancer SCC-4 cells can successfully reverse high meth- ylation status of RUNX3 gene, restore the expression of RUNX3 gene mRNA and protein, increase p53 protein and therefore increase apoptosis.