大豆异黄酮对乳腺癌细胞抑癌基因启动子区的去甲基化作用研究

Reactivation of methylation-silenced tumor suppressor gene E-cadherin by genistein in human breast cancer cells

目的:探讨大豆异黄酮是否能够逆转乳腺癌细胞中关键抑癌基因—上皮型钙粘蛋白基因(E-cadherin)启动子的异常甲基化状态,并有效地重新激活该基因的表达,从而抑制乳腺肿瘤细胞生长。方法:采用不同浓度大豆异黄酮处理乳腺癌SKBR3细胞6d后,通过噻唑蓝比色法检测大豆异黄酮对乳腺癌细胞增殖的影响,进而通过甲基化特异性聚合酶链反应结合半定量PCR方法,检测乳腺癌细胞中E-cadherin基因启动子区CpG岛的去甲基化状态及恢复表达情况。结果:大豆异黄酮在10,20,40,80,160μmol/L浓度对乳腺癌细胞的抑制率分别为(7±2.11)%,(10±3.23)%,(44.33±4.93)%,(84.33±3.06)%,(86.33±3.51)%,其对细胞的生长抑制呈剂量依赖效应。甲基化特异性PCR及半定量PCR结果提示大豆异黄酮对E-cadherin基因启动子区异常甲基化具有去甲基化效应,并能使Ecadherin基因部分恢复表达。结论:植物雌激素大豆异黄酮作为一种天然的表观遗传修饰剂能够通过逆转关键抑癌基因启动子甲基化,激活抑癌基因,抑制乳腺癌细胞的生长,在乳腺肿瘤的预防和治疗中发挥重要作用。

Objective： To explore whether the genistein may reactivate methylation silenced E-cadherin gene in human breast cancer cells and then inhibit the growth of breast cancer cells. Methods.. After 6 days of exposure to various concentrations of genistein （10, 20, 50, 100μM） ,the effects of genistein on the growth of SKBR3 cells were measured by the MTT assay. The CpG island methylation status of E-cadherin was analyzed by methylation-specific PCR （MSP）. And the expression level of E-cadherin mRNA was detected by reverse transcriptase-PCR （RT-PCR）. Results： The MTT results indicated that genistein substantially inhibited the growth of human breast carcinoma cells in a dose-de pendent manner. The inhibitory rates of different treated group （10μM, 20μM, 40μM, 80μM, 160μM） were （7 ±2.11）%, （10±3.23）%, （44.33±4.93）%0, （84.33±3.06） %, （86.33±3.51）%, respectively. MSP and RT-PCR results indicated that genistein reversed SKBR3 CpG island hypermethylation and restored its transcription in SKBR3 cell lines. Conclusion： The results suggest that genistein may serve as natural effective epigenetic modifiers via reactivating the methylation silenced key tumor repressor gene and restoring its expression. And genistein may act as natural effective epigenetic modifiers in the prevention or treatment of cancer.