巢式PCR法检测肝母细胞瘤中p15和p16基因启动子异常甲基化

Detection of abnormal methylation of p15 and p16 gene promotor by nested PCR in hepatoblastoma

目的DNA甲基化被认为是反映细胞内DNA转录状态的重要遗传学标记。该研究旨在对小儿肝母细胞瘤中p15和p16基因5′启动子区CpG岛异常甲基化的相关性进行评估,并分析其与小儿肝母细胞瘤发生发展的关系。方法采用甲基化特异性PCR法,对30例小儿肝母细胞瘤、癌旁和远癌正常组织进行巢式PCR扩增,经凝胶电泳和测序方法检测目的片段。结果30例小儿肝母细胞瘤组织中p15和p16基因5′CpG岛有30%(9/30)和67%(20/30)、癌旁组织中有20%(6/30)和57%(17/30)、远癌正常组织有13%(4/30)和33%(10/30)异常甲基化。在肝母细胞瘤中发现1例p15E2纯合缺失,缺失率为3%(1/30),E1和部分I1区域未见缺失;p16E2和部分I2区域中3例杂合缺失,缺失率为10%(3/30),E1和部分I1区域未见缺失。结论p15和p16基因5′启动子区CpG岛的异常甲基化可能在肝母细胞瘤发生发展中扮演了重要角色,其主要机制可能是启动子区CpG岛甲基化抑制了基因的转录。

Objective DNA methylation has been regarded as an important genetics marker reflecting the transcription state of DNA in cells. This study aimed to assess the methylation of CpG island in 5′ promotor region of p15 and p16 genes in children with hepatoblastoma, and to explore its relationship with the development of hepatoblastoma, Methods The methylation-specific polymerase chain reaction （MSP） and PCR-SSCP techniques were used to analyze DNA methylation. The tumor tissues, the tissues beside the tumor and normal tissues beyond the tumor in 30 cases with hepatoblastoma were studied by nested MSP, The target fragment was verified by gel electrophoresis and sequencing. Results The incidence of abnormal methylation of 5′ CpG island of p15 and pl6 genes in tumor tissues was 30% （9/30） and 67% （20/30） respectively, It was 20% （6/30） and 57% （17/30） respectively in the tissues beside tumor and that was 13% （4/30） and 33% （10/30） respectively in the normal tissues beyond the tumor. Homozygous deletion of exon 2 of p15 gene was found in 1 of 30 cases （3%） in hepatoblastoma, No deletion of exon 1 or intron 1 was found. Heterozygosis deletion of exon 2 of pl6 gene and partial intron 2 was found in 3 of 30 cases （ 10% ）. No deletion of exon 1 or intron 1 was found, Conclusions The abnormal methylation of CpG island in 5′ promotor region of p15 and pl6 genes appears to play a role in the development of hepatoblastoma, which might be attributed to the inhibition of gene transcription by abnormal methyahion of CpG island in the promotor region.