5-氮杂-2’-脱氧胞苷对舌癌细胞株SCC-4细胞RECK基因甲基化水平及侵袭的影响

目的 探讨甲基化酶抑制剂5-氮杂-2′-脱氧胞苷5-aza-2-deoxycytidine (5-aza-Dc) 对舌癌细胞株SCC-4中抑癌基因RECK基因甲基化水平及侵袭能力的影响。方法 用不同浓度的5-aza-Dc处理舌癌细胞株SCC-4,72 h后,以甲基化特异性PCR（MSP）检测SCC-4细胞RECK基因的甲基化状态,实时定量 PCR技术检测SCC-4细胞RECK基因mRNA的表达,Western 印迹检测蛋白质表达,Transwell体外侵袭实验检测侵袭能力。采用SPSS13.0软件包对数据进行统计学分析。结果 MSP检测未处理组细胞RECK基因呈高甲基化状态,经5-aza-dC处理后甲基化得到逆转。实时定量 PCR检测显示,不同浓度5-aza-dC处理SCC-4细胞72 h后, 相对mRNA表达随着药物浓度增加而增加(P<0.05)。Western 印记分析结果显示,不同浓度5-aza-dC组的RECK蛋白表达相对水平随药物浓度增加而增加,SCC-4细胞的侵袭力随药物浓度增加而降低。结论 5-aza-dC可逆转舌癌SCC-4细胞中RECK基因的高甲基化状态,并恢复RECK基因mRNA及蛋白的表达,降低其体外侵袭能力。

Effects of 5-aza-2-deoxycytidine on methylation status of RECK gene and cancer cell invasion in tongue cancer SCC-4 cells

PURPOSE: To investigate the effects of 5-aza-2-deoxycytidine on methylation status and invasion ability of RECK gene in tongue cancer SCC-4 cells. METHODS: Tongue cancer cell line SCC-4 cells were treated with 5-aza-dC at different concentrations for 72 h. Methylation status of RECK gene of SCC-4 cells was detected by methylation specific PCR (MSP), the expression of RECK gene mRNA was detected by real-time quantitative PCR. The expression of RECK protein was detected by Western blot, and the invasion ability of SCC-4 cell was examined by Transwell assay. SPSS13.0 software package was used for statistical analysis. RESULTS: RECK gene of SCC-4 cells was in high methylation status in untreated group, abnormal methylation was effectively reversed by 5-aza-dC treatment. After treatment with different concentration of 5-aza-dC for 72 h, relative mRNA expression level increased gradually (P<0.05). The relative expression level of RECK protein in 5-aza-dC treated group was significantly higher than that in the control group,the invasion ability of SCC-4 cell was decreased gradually. CONCLUSIONS: 5-aza-dC treatment for tongue cancer SCC-4 cells can successfully reverse high methylation status of RECK gene and restore the expression of RECK gene mRNA and protein, and reduced the invasion ability.