姜黄素对鼻咽癌细胞系CNE-1RECK基因表达及甲基化的影响

Effect of curcumin on RECK expression and methylation in nasopharyngeal carcinoma cells CNE-1

目的 探讨姜黄素对鼻咽癌细胞RECK基因表达及甲基化的影响，并探讨可能的机制。方法 体外培养鼻咽癌细胞系CNE-1，用1，10和30μmol/L 姜黄素处理后，用Western blot和实时定量PCR分别检测RECK基因以及DNA甲基化酶1（DNMT1）蛋白和mRNA表达；高效液相色谱-电喷雾质谱检测RECK甲基化，同时用MTT检测CNE-1细胞增殖，EMSA法检测核转录因子Sp1的DNA结合活性。结果 CNE-1细胞未刺激时RECK表达水平较低，而经1-30μmol/L 姜黄素处理后，能显著增强RECK蛋白和mRNA的表达（P〈0.05）。30μmol/L 姜黄素处理CNE-1细胞后， RECK启动子甲基化水平降低至31% （P〈0.05），全基因组甲基化水平降低39% （P〈0.05），细胞核的甲基化活性减少了71.6% （P〈0.05）。同时，姜黄素能显著降低CNE-1细胞中DNMT1蛋白和mRNA表达 （P〈0.05），也能减低CNE-1细胞的存活率 （P〈0.05）。此外， EMSA结果显示，姜黄素处理后，CNE-1细胞中Sp1的DNA活性显著降低。结论 姜黄素可能通过能上调RECK基因表达，降低细胞内甲基化水平，从而可能发挥对CNE-1细胞生长。

Objective To investigate the effect of curcumin on reversion-indueing-cysteine-rich protein with kazal motifs （RECK） gene expression and methylation, and then to analyze potential mechanism. Methods Nasopharyngeal carcinoma cell line CNE-1 was cultured, and stimulated by 1, 10 and 30 μmol/L curcumin for 48 h, expression of RECK and DNA methyltransferases 1 （DNMT1） gene mRNA and protein were examined by Western blot and real-time PCR respectively. RECK methylation was determined by HPLC chromatographic and mass spectrometric methods. Cell proliferation was assessed by MTT assay, and the DNA-binding activity of nuclear factor Spl was detected by EMSA. Results The expression level of RECK was very low in unstimulated cells, and 1 - 30 μmol/L could decrease the protein and mRNA level （ P 〈 0. 05 ）. 30 μmol/L of curcumin could decrease the promoter methylation level to 31% of the basal level （P 〈 0. 05）, and the global DNA methylation level and the methylation activity of the nuclear extract were also decreased about 39% （P 〈 0. 05 ） and 72% （P 〈 0. 05 ）,respectively. Western blot showed that curcumin may also decrease the protein and mRNA level of DNMT1 （P 〈 0. 05 ） and survival rate （P 〈0. 05 ）. In addition, the DNA-binding activity of Spl decreased after curcumin treat- ment. Conclusions Curcumin may inhibit CNE-1 cells growth by up-regulation of RECK gene expression and down-regulation of its methylation.