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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。
AIM: To investigate the effect of 5-aza-2-deoxycytidine (5-aza-Dc), a methylase inhibitor, on the expression of the tumor suppressor gene RECK gene in SCC-4 tongue squamous cell carcinoma The level of impact and the impact of invasion. Methods: The tongue cancer cell line SCC-4 was treated with different concentrations of 5-aza-Dc for 72 h. Methylation-specific PCR (MSP) was used to detect the methylation status of RECK gene in SCC-4 cells. Real- The expression of RECK mRNA in SCC-4 cells was detected by Western blot, and the invasiveness was evaluated by Transwell in vitro invasion assay. SPSS13.0 software package for statistical analysis of the data. Results: RECK gene in untreated MSP cells was hypermethylated and methylated by 5-aza-dC was reversed. Real-time quantitative PCR showed that the relative mRNA expression of SCC-4 cells treated with different concentrations of 5-aza-dC for 72 h increased with the increase of drug concentration (P <0.05). Western blot analysis showed that the relative level of RECK protein expression in 5-aza-dC group increased with the increase of drug concentration, while invasiveness of SCC-4 cells decreased with the increase of drug concentration. CONCLUSION: 5-aza-dC reverses the hypermethylation status of RECK gene in tongue cancer SCC-4 cells and restores the expression of RECK mRNA and protein, and reduces the invasiveness of RECC gene in vitro.