[CpG岛甲基化状态对荧光素酶报告基因检测结果的影响]

[Effects of methylation status of CpG islands on results of luciferase reporter assay].

作者信息

Zhang Bao-Zhen, Deng Da-Jun

机构信息

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University School of Oncology, Beijing Cancer Hospital and Institute, Beijing, China.

出版信息

Zhonghua Yu Fang Yi Xue Za Zhi. 2009 Jul;43(7):601-6.

PMID:19954072

Abstract

OBJECTIVE

To investigate the effects of methylation status of CpG islands of endogenous E-cadherin (CDH1) gene on the promoter activity of corresponding genes in reporter assays.

METHODS

The methylation statuses of CpG island of CDH1 in 8 different cell lines were detected by methylation-specific PCR. CDH1 protein was analyzed by Western blotting. Two sets of pGL3 reporter vectors with different genotypes/haplotypes of the CDH1 promoter were constructed [ pGL3-A(-73)/-C(-73) pGL3-H1/-H4] and used to transfect these cell lines. The differences between these promoter reporter vectors were analyzed by t-test.

RESULTS

(1) CDH1 CpG island was unmethylated in AGS, MCF7, MKN74, and PC-3 cell lines, expressed in MCF7, MKN74, and PC-3, but not in AGS. Expression of CDH1 was silenced by methylation in HeLa, BGC823, A549, and RKO cell lines. (2) In the four CDH1-unmethylated MCF7, MKN74, PC-3, and AGS cell lines, the promoter activities of pGL3-C(-73), (as 0.78 +/- 0.10, 0.17 +/- 0.01, 0.11 +/- 0.01, 1.19 +/- 0.18) were significantly higher than those of pGL3-A(-73) (as 0.30 +/- 0.08, 0.07 +/- 0.01, 0.07 +/- 0.01, 0.39 +/- 0.04) (t values are -6.298, - 12.349, -8.128, -7.388, and P <. 0.1). However, in the four CDH1-methylated HeLa, BGC823, A549, and RKO cell lines, the promoter activity of pGL3-C(-73) (as 0.09 +/- 0.02, 0.13 +/- 0.02, 0.05 +/- 0.01, 0.01 +/- 0.00) was significantly lower than that of pGL3-A(-73) (as 0.16 +/- 0.01, 0.25 +/- 0.01, 0.11 +/- 0.03, 0.03 +/- 0.00) (t valued at 5.958, 11.189, 3.661, 13.866, and P<0.05). (3) In the unmethylated MKN74 and methylated RKO cell lines, the promoter activities of pGL3-H1/-H4 were obviously and contrarily different (as 1.57 +/- 0.23/0.94 +/- 0.06 and 0.38 +/- 0.02/0.50 +/- 0.04, t values were 4.577 and -4.915, P values were 0.010 and 0.003).

CONCLUSION

The methylation status of CpG island of the target gene in the tested cell lines affects the promoter activity in Reporter Assay significantly. The most active one may be the most suppressive one.

摘要

目的

在报告基因检测中研究内源性E-钙黏蛋白（CDH1）基因CpG岛甲基化状态对相应基因启动子活性的影响。

方法

采用甲基化特异性PCR检测8种不同细胞系中CDH1基因CpG岛的甲基化状态。通过蛋白质免疫印迹法分析CDH1蛋白。构建两组具有不同基因型/单倍型的CDH1启动子的pGL3报告载体[pGL3-A(-73)/-C(-73)、pGL3-H1/-H4]，并用于转染这些细胞系。采用t检验分析这些启动子报告载体之间的差异。

结果

（1）在AGS、MCF7、MKN74和PC-3细胞系中，CDH1基因CpG岛未甲基化，在MCF7、MKN74和PC-3细胞系中表达，但在AGS细胞系中不表达。在HeLa、BGC823、A549和RKO细胞系中，CDH1基因的表达因甲基化而沉默。（2）在四个CDH1基因未甲基化的MCF7、MKN74、PC-3和AGS细胞系中，pGL3-C(-73)的启动子活性（分别为0.78±0.10、0.17±0.01、0.11±0.01、1.19±0.18）显著高于pGL3-A(-73)的启动子活性（分别为0.30±0.08、0.07±0.01、0.07±0.01、0.39±0.04）（t值分别为-6.298、-12.349、-8.128、-7.388，P<0.01）。然而，在四个CDH1基因甲基化的HeLa、BGC823、A549和RKO细胞系中，pGL3-C(-73)的启动子活性（分别为0.09±0.02、0.13±0.02、0.05±0.01、0.01±0.00）显著低于pGL3-A(-73)的启动子活性（分别为0.16±0.01、0.25±0.01、0.11±0.03、0.03±0.00）（t值分别为5.958、11.189、3.661、13.866，P<0.05）。（3）在未甲基化的MKN74细胞系和甲基化的RKO细胞系中，pGL3-H1/-H4的启动子活性明显相反（分别为1.57±0.23/0.94±0.06和0.38±0.。02/0.50±0.04，t值分别为4.577和-4.915，P值分别为0.010和0.003）。