5-氮杂胞苷对甲状腺乳头状癌细胞TPC-1中Dickkopf同源序列3基因去甲基化的转录调节作用

Effect of 5-azacytidine on growth of thyroid papillary cancer-1 cell line and expression of the dick-kopf homoloR 3 Rene

目的观察甲基化抑制剂5一氮杂胞苷（5-azaC）对甲状腺乳头状癌细胞TPC一1生长的抑制作用及对抑癌基因Dickkopf同源序列3（DDK3）的转录调节作用。方法经2、5、10、20、50、100、200、400ixmol／L的5-azaC处理TPC一1细胞后，采用噻唑蓝（MTT）观察药物对细胞的生长抑制作用；以逆转录一聚合酶链反应（RT—PCR）、甲基化特异性PCR（MSP）检测细胞经5、10、20、50μmol／L的5-azaC处理后DKK3基因mRNA的表达及DKK3基因甲基化状态。结果经不同浓度5-azaC处理TPC一1细胞后，MTF检测到细胞的生长受到抑制，且呈时间和剂量依赖性（P〈0．05）；经5、10、20、50μmol／L的5-azaC处理48h后，DKK3mRNA相对表达量分别为0．208±0．017、0．365±0．013、0．489±0．017、0．582±0．011，表达强度与5-azaC浓度呈剂量依赖关系（F=370．356，P〈0．01）；MSP检测结果显示，经5-azaC处理后，DKK3基冈启动子高甲基化的区域发生去甲基化．、结论5-azaC能有效逆转TPC—l甲状腺乳头状癌细胞DKK3基因的异常甲基化，从而激活DKK3基因表达，抑制肿瘤细胞生长。

Objective To study the effects of 5-azacytidine （5-azaC）, a methylation inhibitor, on the growth of thyroid papillary cancer-1 （ TPC-1 ） papillary thyroid carcinoma cells and the transcription regulation of DNA guanine cytosine-phosphoric acid-motif （CpG） island demethylation on dickkopf homolog 3 （ DKK3 ） tumor suppressor gene. Methods Methyl thiazol tetrazolium （MTF） method was used to detect the growth of TPC-1 cells after treated with 5-azaC （2, 5, 10, 20, 50, 100, 200 and 400 ~mol/L）. The expression of DKK3 mRNA and methylation status were examined by using methylation-specific polymerase chain reaction （MSP） and reverse transcription-polymerase chain reaction （RT-PCR） after TPC-1 cells treated with 5-azaC （5, 10, 20 and 50 I^mol/L）. Results 5-azaC reduced viability of TPC-1 ceils in a dose- and time-dependent manner （P 〈 0. 05）. After TPC-1 cells were treated with 5-azaC （5, 10, 20 and 50 μmol/L） for 48 h, the relative expression of DKK3 mRNA was 0. 208 ±0. 017, 0. 365± 0. 013, 0. 489±0. 017 and 0. 582±0. 011 respectively, which was gradually increased in a dose-dependent manner （ F = 370. 356, P 〈 0.05 ）. Meanwhile, DKK3 gene was demethylated partly in TPC-1 cells treated with 5-azaC. Conclusion 5-azaC might effectively cause the demethylation of DKK3 gene CpG-rich promoter regions, which may activate the expression of DKK3 gene and inhibit the growth of TPC-1 cells.