结肠癌细胞CDH1基因启动子甲基化对E-上皮钙黏素和β-连接素表达的调控作用

Regulation of CDH1 gene promoter methylation on expression of E-cadherin and β-catenin in human colon carcinoma ceils

目的观察结肠癌细胞中上皮钙黏素基因（CDH1）启动子甲基化对上皮钙黏素（E—cadherin）和β-连接素（β—catenin）表达的影响。方法通过甲基化特异性PCR（MSP）、逆转录-聚合酶链反应（RT—PCR）检测DNA甲基化抑制剂5-氮-2′-脱氧胞苷（5-Aza-CdR）干预前后人结肠癌HT-29细胞株CDH1基因启动子甲基化状态及E—cadherin mRNA的改变，并运用免疫荧光标记及激光共聚焦观察E—cadherin和β-catenin在细胞内的表达及分布。结果（1）HT-29细胞在用药前CDH1基因启动子甲基化扩增阳性，非甲基化扩增阴性，用5-Aza-CdR处理后CDH1基因启动子甲基化扩增阴性，非甲基化扩增阳性；（2）5-Aza—CdR处理前细胞RT—PCR不能扩增出E—cadherin mRNA特异性条带，5-Aza—CdR处理后则可检测到E—cadherinmRNA转录，且mRNA水平与药物的浓度呈正相关，平均灰度比值1μmol／L时为0．491±0．011，2μmol／L时为0．568±0．013（P〈0．05：（3）5-Aza-CdR处理前细胞未见E-cadherin表达，13-catenin主要表达于细胞质及细胞核中，5-Aza—CdR处理后在细胞膜可见E—cadherin及-catenin表达。且随着5-Aza—CdR诱导E-cadherin的表达，β-catenin在细胞膜的分布增加，而在细胞质及细胞核的分布明显减少。免疫荧光双重染色显示β-catenin胞膜分布与E—cadherin一致。结论CDH1基因启动子甲基化可能是导致结肠癌细胞E—cadherin表达缺失及β—catenin异位分布的重要因素。

Objective To observe the effects of CDH1 gene promoter methylation on the expres- sion of E-cadherin and β-catenin in human colon carcinoma cells. Methods Methylation specific PCR （MSP） and reverse transcription-polymerase chain reaction （RT-PCR） methods were utilized to examine methylation status of CDH1 gene promoter and the changes of E-cadherin mRNA in colon carcinoma cell line HT-29 before and after the treatment with 5-Aza-CdR. The expression of E-cadherin and localization of β-catenin were labeled by immunofluorescence and analyzed under a laser scanning confocal microscope. Results （1） CDH1 gene promoter methylation was positive and unmethylation was negative in HT-29 cells before the treatment with 5-Aza-CdR. After treatment with 5-Aza-CdR for 24 h, methylation turned negative and unmethylation was detected; （2） The E-cadherin mRNA failed to be amplified in cells, whereas it could be detected after the treatment. The mRNA expression level of E-cadherin expressed as average gray ratio was 0. 491 ± 0. 011 and 0. 568 ±0. 013 respectively after treatment with 1 and 2 μmoL/L 5-Aza-CdR （P 〈 0. 05 ; （3） Before treatment with 5-Aza-CdR, the expression of E-cadherin was not detected and β-catenin was detected in cytoplasm and nucleus, while both E-cadherin and β-catenin staining was positive on the cell membrane by immunofluorescence after the treatment. With the expression of E-cadherin induced by 5-Aza-CdR, the membranous expression of β-catenin was elevated and nuclear expression of β-catenin was reduced. Immunofuorescence double staining displayed the distribution of β-catenin was corresponded with E-cadherin on the cell membrane. Conclusion CDH1 gene promoter methylation may lead to the loss of E-eadherin expression and the altered distribution of β-eatenin in colon carcinoma cells.