5-氮-2’-脱氧胞苷诱导E-cadherin基因去甲基化及转录和表达  

The impact of 5-Aza-CdR on demathylation of transcription regulation of E-cadherin gene in tumor cell line

作　　者：郑本波[1] 何文智[2] 王锦红[2] 何永林[1] 

机构地区：[1]四川德阳市人民医院普外一科,四川德阳618000 [2]四川大学分子生物学教研室,四川成都610044

出　　处：《中国癌症杂志》2006年第9期744-747,共4页China Oncology

摘　　要：背景与目的用去甲基化药物5-氮-2’-脱氧胞苷(5-Aza-CdR)对细胞株MDA-MB-435抑癌基因E-cadherin(上皮钙粘附素,简称E-cad)表达的影响。观察DNA甲基化的可逆性,为肿瘤治疗提供新的靶点。方法在用5-Aza-CdR处理MDA-MB-435细胞株前、后分别应用甲基化特异性PCR(MSP)检测E-cad基因5’-CpG岛甲基化改变;用免疫组化(LsAB法)检测E-cad蛋白表达;用半定量RT-PCR观察E-cadmRNA的变化。结果①MDA-MB-435细胞在用药前E-cad基因甲基化PCR扩增阳性(116bp),而非甲基化PCR扩增阴性。用0.5μmol/L的5-Aza-CdR处理后发现该细胞E-cad基因甲基化PCR扩增阴性,而非甲基化PCR扩增出阳性条带(97bp)。②免疫细胞化学法检测E-cad蛋白用药前细胞未见E-cad蛋白表达。用5-Aza-CdR处理后在细胞膜可见E-cad染色阳性。③RT-PCR发现用5-Aza-CdR处理前细胞不能扩增出630bp的E-cadmRNA特异性条带;用0.5、1.0、2.0、5.0μmol/L的5-Aza处理细胞后都可检测到E-cadmRNA转录,且mRNA水平与药物的浓度呈正相关。结论去甲基化药物5-Aza-CdR能有效地逆转MDA-MB-435细胞株的E-cad基因异常甲基化,诱导mRNA转录和蛋白的表达。Background and purpose： To explore the possibility of genetic re-expression silenced by DNA aberrant hypermethylation which is a common epigenetic modification in carcinogenesis. 5-Aza-CdR, an inhibitor of DNA metbylation, was used to determine the effects of expression of tumor suppressor gene E-cadherin in tumor cell lines. Methods： Methylation specific PCR（MSP） was utilized to examine methylation status of E-cad gene on breast carcinoma cell line MDA-MB-435 before and after the treatment with 5-Aza-CdR. Immunohistochemistry（IHC） was used to test the expression of E-cad protein. Semi-quantitative RT-PCR method was used to detect the changes of E-cad mRNA. Results： 1）. E-cad methylation was positive（116bp） and unmethylation was negative on MDA-MB-435 cell before the treatment with 5-Aza- CdR. After being treated with 5. Oumol/L 5-Aza-CdR for 3 days, methylation turned negative and unmethylation positive bands（97bp） were detected. 2）. The E-cad protein expression was not detected by immunohistochemistry on MDA-MB-435 cell before the treatment, while E-cad staining was positive on the cell membrane after the treatment. 3）. The E-cad mR- NA failed to be amplified in cells before the treatment. After incubation at variable concentrations of 0. 5 μmol/L, 1.0 μmol/L, 2.0 μmol/L and 5.0 μmol/L 5-Aza-CdR for 3 days, respectively, E-cad mRNA expression was detected on the fourth day in a dose-dependent manner. Correlation between the mRNA expression level and the agent concentration was observed. Conclusions： The demethylation agent 5-Aza-CdR can reverse the aberrant E-cad methylation status in MDA-MB- 435 and re-expressed E-cad mRNA and protein.

关 键 词：甲基化 甲基化特异性PCR 基因 上皮钙粘附素 

分 类 号：R73-361[医药卫生—肿瘤]