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目的 DLC1下调与多种肿瘤的发生相关,DLC1基因启动子区CpG岛甲基化可能是其基因表达下调或缺失的重要机制。本研究探讨姜黄素对MDA-MB-361人乳腺癌细胞株DLC1基因表达的影响及其机制。方法应用甲基化特异性聚合酶链反应(methylation specific polymerase chain reaction,MSP)、逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)及量子点免疫荧光细胞化学(quantum dots immunofluorescence cytochemistry,QDICC)检测不同浓度姜黄素(10、20、40μmol/L)处理乳腺癌细胞株MDA-MB-361前后DLC1基因启动子甲基化状态、DLC1mRNA及蛋白表达水平变化。结果 MDA-MB-361细胞DLC1基因启动子区CpG岛呈甲基化状态,10μmol/L姜黄素无去甲基化作用,无DLC1mRNA和蛋白表达。经过20和40μmol/L姜黄素处理后,DLC1基因启动子区呈现一定程度的去甲基化,其mRNA和蛋白表达增加,mRNA相对表达量分别为0.88±0.14和1.31±0.11,DLC1蛋白均(++)。不同浓度姜黄素处理组间差异有统计学意义,F=157.344,P<0.001。40μmol/L姜黄素较20μmol/L姜黄素处理细胞后mRNA和蛋白表达稍增加,但差异无统计学意义,F=16.325,P=0.016。结论 MDA-MB-361乳腺癌细胞中DLC1基因表达沉默与启动子甲基化有关,20和40μmol/L姜黄素能部分逆转该细胞的DLC1基因甲基化状态,恢复该基因表达。
Purpose DLC1 down-regulation is associated with a variety of oncogenes. Methylation of CpG islands in the promoter region of DLC1 gene may be an important mechanism of down-regulation or loss of gene expression. This study was to investigate the effect of curcumin on the gene expression of DLC1 in MDA-MB-361 human breast cancer cell line and its mechanism. Methods Methylation-specific polymerase chain reaction (MSP), reverse transcription-polymerase chain reaction (RT-PCR) and quantum dot immunofluorescence cytochemistry Immunofluorescence cytochemistry (QDICC) was used to detect the methylation status of DLC1 gene promoter and the expression of DLC1 mRNA and protein in breast cancer cell line MDA-MB-361 after treated with different concentrations of curcumin (10, 20 and 40 μmol / L) Results The CpG island in the promoter region of DLC1 gene in MDA-MB-361 cells was methylated. There was no demethylation of 10μmol / L curcumin, but no DLC1 mRNA and protein expression. After treated with 20 and 40 μmol / L curcumin, the promoter region of DLC1 gene was demethylated and the mRNA and protein expressions were increased. The relative mRNA levels of DLC1 gene were 0.88 ± 0.14 and 1.31 ± 0.11, respectively ++). Curcumin at different concentrations had significant difference between the two groups (F = 157.344, P <0.001.40μmol / L). Curcumin slightly increased mRNA and protein expression compared with those treated with 20μmol / L curcumin, but the difference was not significant = 16.325, P = 0.016. Conclusion Silencing of DLC1 gene in MDA-MB-361 breast cancer cells is related to promoter methylation. Curcumin at 20 and 40 μmol / L partially reversed the methylation status of DLC1 gene and restored the gene expression.