人肝癌细胞遗传印记基因PEG10印记状态不受DMR甲基化调控

The imprinting status of PEG10 is not regulated by the methylation of differentially methylated region in human liver cancer

目的探讨人肝癌细胞遗传印记基因(genetic imprinted gene)PEG10差异性甲基化区(differntially DNA methylated region,DMR)甲基化状态在其印记调控中的作用。方法以单核苷酸多态性(single nucleotide polymorphisms,SNP)位点为等位基因标记,分析人肝癌HepG2细胞PEG10印记状态;采用RT-PCR、免疫组化及Western-blot检测PEG10表达水平;重亚硫酸盐修饰DNA测序法分析PEG10-DMR甲基化状态;再应用甲基基团供体S-腺苷蛋氨酸(S-adenosyl-L-methionine,SAM)及DNA甲基转移酶抑制剂5-氮杂胞苷(5-azacytidine,5-azaC)在体外及荷瘤裸鼠体内调控HepG2细胞PEG10-DMR甲基化状态,观察PEG10-DMR甲基化状态改变对PEG10印记状态及表达水平的影响。结果遗传印记基因PEG10在HepG2细胞呈双等位基因表达的印记丢失。与正常肝细胞HL7702相比,其PEG10-DMR甲基化水平显著升高。调控PEG10-DMR甲基化水平可改变PEG10的表达水平,但对其印记状态无影响。结论 PEG10-DMR甲基化不参与人肝癌细胞PEG10的印记调控,但可调控其表达水平。

Objective To investigate the role of the methylation of differntially DNA methylated region of PEG10 in the regulation of its imprinting status in human liver cancer cells. Methods Single nucleotide polymorphisms （SNP） were regarded as marker between two alleles to analyze the imprinting syatus of PEG10. RT-PCR, immunohistochemistry staining and Western-blot analysis were used to examine the expression of PEG10. Bisulfite sequencing was used to ana- lyze the methylation of PEG10- DMR. Cells were treated with the methyl donor S-adenosyl-L-methionine （SAM） or the DNA methyltransferase inhibitor 5-azacytidine （5-azaC） , and then imprinting status and PEG10 expression levels were observed in vitro and in vivo. Results PEG10 was biallele expression in HepG2, that was loss of imprinting. PEG10- DMR methylation levels were significantly higher in HepG2 cells than that in HL7702 cells. PEG10- DMR methylation regulated the expression levels of PEG10, but had no effect on the imprinting status. Conclusion PEG10-DMR methylation is involved in the regulation of expression but not imprinting in human liver cancer cells.