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首页> 外文期刊>Journal of Huazhong University of Science and Technology >Re-expression of RASSF1A by 5-Aza-CdR Induced Demethylation of the Promoter Region in Human Biliary Tract Carcinoma Cells
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Re-expression of RASSF1A by 5-Aza-CdR Induced Demethylation of the Promoter Region in Human Biliary Tract Carcinoma Cells

机译：5-Aza-CdR诱导人胆道癌细胞中启动子区去甲基化的RASSF1A重新表达。

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Hypermethylation of the promoter region is an important mean for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferase are considered to be the main cause of promoter hypermethylation. In order to further explore the epigenetic mechanism of tumor suppressor gene RASSF1A inactivation, 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, was used to treat the human biliary tract carcinoma cell line QBC-939 at the concentration of 5 μmol/L for 24 h in this study. After the chemical intervention with 5-Aza-CdR, the methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression alteration of RASSF1A mRNA and protein were observed by RT-PCR and Western Blot respectively. Following the treatment with 5-Aza-CdR, methylaiton status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1 FA expression at transcriptional level and a 40 kDa (1kDa=0.9921 ku) protein band which represented RASSF1A expression at protein level were detected by RT-PCR and Western Blot respectively in the experimental group cells and there were no corresponding bands in the control group cells. The experimental results suggest that 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by DNA methylation and induce the re-expression of RASSF1A in QBC-939. This study also suggest that the mechanism of RASSF1A inactivation is very closely related to the methylation of the promoter region, which may provide a new epigenetic understanding for tumor related gene inactivation and the pathogenesis of biliary tract carcinoma.
机译：启动子区域的高甲基化是许多癌症相关基因转录抑制的重要手段，并且DNA甲基转移酶的过表达和/或活性增加被认为是启动子高甲基化的主要原因。为了进一步探索抑癌基因RASSF1A失活的表观遗传机制，使用了DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-CdR）来治疗人胆道癌细胞系QBC-939在本研究中，浓度为5μmol/ L持续24小时。用5-Aza-CdR进行化学干预后，通过甲基化特异性PCR（MS-PCR）检测RASSF1A基因启动子区域的甲基化状态，并通过RT-PCR和Western观察到RASSF1A mRNA和蛋白的表达变化。分别吸干。用5-Aza-CdR处理后，RASSF1A基因启动子区域中的甲基质子状态从甲基化反转为未甲基化。在实验组细胞中分别通过RT-PCR和Western Blot检测到一个在转录水平上代表RASS1 FA表达的280 bp DNA条带和一个在蛋白水平上代表RASSF1A表达的40 kDa（1kDa = 0.9921 ku）蛋白带。对照组细胞中没有相应的条带。实验结果表明5-Aza-CdR可以在RASSF1A的启动子区域诱导去甲基化。它还可以逆转由DNA甲基化引起的表观遗传转录沉默，并诱导QBC-939中RASSF1A的重新表达。这项研究还表明，RASSF1A失活的机制与启动子区域的甲基化密切相关，这可能为肿瘤相关基因失活和胆道癌的发病机制提供新的表观遗传学认识。

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