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目的探讨莱菔硫烷(SFN)对鼻咽癌CNE-2细胞RASSF1A甲基化及蛋白表达水平的影响。方法体外培养鼻咽癌CNE-2细胞,用0,5、10、30μM的SFN与其孵育24～96h,用MTT法检测细胞的增值情况;流式细胞术分析细胞周期的改变;采用甲基化特异性PCR检测RASSF1A基因启动子区域的甲基化改变情况;提取SFN处理后细胞的RNA,用逆转录PCR检测RASSF1A的表达水平,并用Western blot检测处理前后RASSF1A蛋白的表达。结果 SFN能以时间和剂量依赖性方式抑制CNE-2细胞增值,并能使细胞周期阻滞于S期和G2/M期。随着SNF浓度的增加,甲基化RASSF1A水平逐渐减弱,非甲基化RASSF1A水平逐渐增多。同时SFN能明显上调RASSF1A的mRNA和蛋白表达水平。结论 SFN能使RASSF1A基因去甲基化,并上调非甲基化基因的表达水平,从而抑制细胞的增值和分化而具有抗肿瘤的效应。
Objective To investigate the effect of sulforaphane (SFN) on methylation and protein expression of RASSF1A in nasopharyngeal carcinoma CNE-2 cells. Methods Nasopharyngeal carcinoma CNE-2 cells were cultured in vitro, incubated with SFN at 0, 5, 10 and 30μM for 24-96 h. Cell proliferation was measured by MTT assay. Cell cycle was analyzed by flow cytometry. Methylation The methylation of RASSF1A promoter region was detected by specific PCR. The RNA of SFN-treated cells was extracted. The expression of RASSF1A was detected by reverse transcription polymerase chain reaction (RT-PCR). The protein expression of RASSF1A was detected by Western blot. Results SFN inhibited the proliferation of CNE-2 cells in a time and dose-dependent manner and blocked the cell cycle in S phase and G2 / M phase. With the increase of SNF concentration, the level of methylated RASSF1A gradually decreased and the level of unmethylated RASSF1A gradually increased. At the same time, SFN could up-regulate the mRNA and protein expression of RASSF1A. Conclusion SFN can demethylate RASSF1A gene and up-regulate the expression of non-methylated genes, thereby inhibiting the proliferation and differentiation of cells and having anti-tumor effect.