Expression and LOI of Igf2 in mouse tumors

IGF2在胎儿期在小鼠的大部分组织中都有表达，而在成年小鼠中，除大脑外，IGF2的表达受到抑制。IGF2是一个印记基因，其父系等位基因表达，而其物质等位基因被抑制。推测Igf2的印迹是通过Igf2基因内特定区域的甲基化以及位于Igf2下游的H19基因的甲基化来维持的。Igf2基因由6个外显子组成，lgf2基因包括外显子1-3和外显子4-6中的一个。另一方面，由于大多数小鼠肿瘤表达Igf2，而且由于肿瘤偶尔在Igf2转基因小鼠中发展，因此Igf2被认为在肿瘤的发生中起到了作用。本研究旨在研究小鼠肿瘤细胞中特异性Igf2基因的表达、基因甲基化和转录起始点。(1)通过分析Igf2外显子6中CA重复序列的多态，发现小鼠肝癌细胞系中存在频繁的印迹丢失(LOI)。(2)肝细胞癌中P1、P2、P3启动子均为活性启动子，而LE细胞中仅有P2、P3启动子活性。(3)在正常肝细胞原代培养中有Igf2的表达，但其印迹在正常肝细胞中保持不变。(4)通过对Igf2上游非编码区的甲基化特异性PCR-DNA测序，鉴定了CpG基因的甲基化状态。

Igf2 is expressed in most tissues during a fetal period in mice, while it is suppressed except for the brain in adult. Igf2 is an imprinted gene of which paternal allele is expressed, while its material allele is suppressed. It is suggested that the imprinting of Igf2 is maintained by methylation of the specific regions within the Igf2 gene as well as methylation of the H19 gene which is located downstream of Igf2. The Igf2 gene contains 6 exons, and lgf2 mRNA includes one of exons 1-3 with exons 4-6 in common. On the other hand, since most mouse tumors express Igf2 and since tumors occasionally develop in Igf2 transgenic mice, Igf2 is thought to have a role in tumor development. A purpose of this study was to investigate the allele specific Igf2 expression, methylation of Igf2 gene and transcription start points in mouse tumor cells.Following points became clear in 1998- 1999.(1) By analyzing the polymorphic CA repeat within Igf2 exon 6, loss of imprinting (LOI) was frequently detected in mouse HCC cell lines. On the other hand, LOI was hardly found in liver epithehal (LE) cells derived from the normal liver.(2) All the P1, P2, P3 promoters were active in the HCC cells, while only P2 and P3 promoters were active in LE cells.(3) Igf2 was expressed in the primary cultures of normal hepatocytes, but its imprinting is maintained in this case.(4) Paternal allele specific methylation of CpG was identified by methylation specific PCR-DNA sequencing in the upstream non-coding region in Igf2.