细胞荧光素酶报告基因试验受CpG岛甲基化状态影响

Effects of methylation status of CpG islands on results of luciferase reporter assay

目的研究细胞内源性上皮型钙黏素基因（E-cadherin，CDH1）启动子CpG岛甲基化状态是否影响报告基因试验的结果。方法甲基化特异性PCR法测定8种人肿瘤细胞系CDH1启动子CpG岛甲基化状态，免疫印迹法测定其蛋白表达；根据该基因启动子-73位A／C多态和单倍体型构建2组长短不同的CDH1启动子-荧光素酶报告基因[pGI3-A（-73]）／-C（-73]）和pGL3-H1／-H4]，活性差异采用t检验进行统计分析。结果（1）CDH1在MCF7、MKN74、PC-3和AGS细胞中CpG岛未甲基化，除AGS外均存在CDH1表达；在HeLa、BGC823、A549和RKO细胞中CpG岛甲基化并且无表达；（2）在CDH1未甲基化细胞系MCF7、MKN74、PC-3和AGS中pGL3-C（-73）报告基因活性（分别为：0．78±0．10、0．17±0．01、0．11±0．01、1．19±0．18）均显著高于pGL3-A（-73）报告基因活性（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值均〈0．01），而在CDH1甲基化细胞中则相反[HeLa、BGC823、A549和RKO细胞中pGL3C（-73）、报告基因活性分别为：0．09±0．02、0．13±0．02、0．05±0．01、0．01±0．00，pGL3-A（-73）活性分别为：0．16±0．01、0．25±0．01、0．11±0．03、0．03±0．00，pGL3-C（-73）活性低于pGL3-A（-73），t值分别为：5．958、11．189、3．661、13．866，P值均〈0．05]；（3）在MKN74和RKO细胞中，pGL3-H1／-H4的启动子活性分别为1．57±0．23／0．94±0．06和0．38±0．02／0．50±0．04，差异亦存在统计学意义（t值为4．577和-4．915，P值为0．010和0．003），并且在两种细胞中活性相反。结论细胞内源性目的基因CpG岛甲基化状态对报告基因试验结果可能有重要影响，高活性基因型的启动子也是容易受抑制的启动子。

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 CDHI 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） 和 pGI3-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 CDHl-unmethylated MCF7, MKN74, PC-3, and AGS cell lines, the promoter activities of pGL3-C（ -73） （ as 0. 78 ± 0. 10, -. 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 〈 0.01 ）. However, in the four CDH1-methylated HeLa, BGC823, A549, and RKO cell lines, the promoter activity of pGI3-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 pGI3-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 pGI3-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.