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目的:探讨甲基化转移酶抑制剂[5-氮杂-2′-脱氧胞苷(5-Aza-dC)]及组蛋白去乙酰化转移酶抑制剂[曲古抑菌素A(TSA)]对喉癌细胞系中抑癌基因RASSF1A甲基化水平及基因表达的影响。方法:5-Aza-dC及TSA处理体外培养的Hep-2细胞,应用Realtime PCR方法及甲基化特异性PCR(MSP)方法分别检测药物干预前后细胞中抑癌基因RASSF1A表达及甲基化情况。结果:①在Hep-2细胞中未经药物干预前抑癌基因RASSF1A表现为甲基化,抑癌基因RASSF1A弱表达。②在5-Aza-dC及TSA的作用下,Hep-2细胞系中RASSF1A基因的甲基化状态得到了逆转。其中5-Aza-dC及TSA联合应用效果与单独应用5-Aza-dC效果相似,单独应用TSA无明显效果。③在5-Aza-dC及TSA的作用下,Hep-2细胞系中RASSF1A基因表达上调,其中5-Aza-dC作用较TSA作用强,2种药物联合应用起协同增效作用。结论:喉癌细胞系中抑癌基因RASSF1A启动子甲基化可能是导致其基因失活的主要原因但并不是惟一原因。应用5-Aza-dC及TSA能够通过逆转抑癌基因RASSF1A的DNA甲基化水平和组蛋白去乙酰化水平从而使其表达得到了增强。
Objective: To investigate the effects of methylated transferase inhibitor 5-Aza-dC and histone deacetylase inhibitor trichostatin A (TSA) ] On the expression of RASSF1A methylation and gene expression in laryngeal carcinoma cell lines. Methods: Hep-2 cells cultured in vitro were treated with 5-Aza-dC and TSA. Realtime PCR and methylation-specific PCR (MSP) were used to detect the expression and methylation of tumor suppressor gene RASSF1A . Results: ① Hep-2 cells without methylation of tumor suppressor gene RASSF1A showed methylation, tumor suppressor gene RASSF1A weakly expressed. ② Under the action of 5-Aza-dC and TSA, the methylation status of RASSF1A gene in Hep-2 cell line was reversed. The combination effect of 5-Aza-dC and TSA was similar to the effect of 5-Aza-dC alone. TSA alone had no significant effect. ③ The expression of RASSF1A gene in Hep-2 cell line was up-regulated by 5-Aza-dC and TSA. The effect of 5-Aza-dC was stronger than that of TSA, and the combination of two drugs played synergistic effect. CONCLUSION: Methylation of the tumor suppressor gene RASSF1A in laryngeal carcinoma cell lines may be the main reason for its inactivation but not the only cause. Application of 5-Aza-dC and TSA enhanced its expression by reversing DNA methylation and histone deacetylation of the tumor suppressor gene RASSF1A.