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目的探讨17-β雌二醇(17-βE2)对人卵巢癌细胞株SKOV3中HOXA10基因表达及基因启动子区CpG岛DNA甲基化的影响。方法体外培养人卵巢癌细胞株SKOV3,按实验目的分别加入1×10-6、1×10-7、1×10-8mol/L的17-βE2培养48h后,收集细胞,分别采用实时荧光定量RT-PCR反应和Westernblot检测HOXA10mRNA和蛋白表达;提取SKOV3细胞基因组DNA,纯化、修饰后用甲基化特异性聚合酶链反应检测HOXA10基因启动子区CpG岛DNA甲基化状态。结果与对照组相比,不同浓度的17-βE2可显著提高SKOV3细胞中HOXA10基因mRNA和蛋白表达(P<0.05)。同时17-βE2作用后HOXA10基因呈部分甲基化状态。结论 17-βE2可以逆转SKOV3细胞中HOXA10基因启动子区CpG岛DNA甲基化状态,使HOXA10基因的表达上调。
Objective To investigate the effects of 17-β estradiol (17-βE2) on HOXA10 gene expression and CpG island DNA methylation in human ovarian cancer cell line SKOV3. Methods Human ovarian cancer cell line SKOV3 was cultured in vitro. The cells were cultured in the presence of 17 × β-E2 at 1 × 10-6, 1 × 10-7 and 1 × 10-8 mol / L respectively for 48 hours. The cells were harvested and quantified by real-time fluorescence quantitative The mRNA and protein expression of HOXA10 were detected by RT-PCR and Western blot. The genomic DNA of SKOV3 cells was extracted and purified. The DNA methylation status of HOXA10 gene promoter CpG island was detected by methylation-specific polymerase chain reaction. Results Compared with the control group, different concentrations of 17-βE2 significantly increased HOXA10 mRNA and protein expression in SKOV3 cells (P <0.05). HOXA10 gene was partially methylated after 17-βE2 treatment. Conclusion 17-βE2 can reverse DNA methylation status of HOXA10 gene promoter region CpG island in SKOV3 cells, and up-regulate HOXA10 gene expression.