The analysis of genomic imprinting in ovarian teratomas and choriocarcinoma

卵巢畸胎瘤和绒毛膜癌的基因组印记分析

• 批准号: 08671888
• 负责人: SIMOYA Kouichirou
• 金额: $ 1.41万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671888/
• 关键词: genomic imprinting; human imprinted genes; IGF2; H19; SNRPN; ovarian choriocarcinoma; HI9; 絨毛癌; 胚細胞系腫瘍; ゲノム・インプリント解除; HLA-G遺伝子; IGF2.H19.SNRPN遺伝子

Several genes have been discovered to be subject to genomic imprinting. They include human genes such as IGF2 and SNRPN,which are expressed exclusively from the paternal allele and H19, which is expressed from the maternal allele in most somatic and extra-embryonic organs. In order to examine the variance of imprinting in ovarian teratomas and to identify the imprinting status, we investigated expression level and the allele-specific expression of three human imprinted genes (IGF2, H19, and SNRPN) in a number of dermoid cysts. We also examined the methylation status of the alleles, where imprint-specific differential methylation has been demonstrated in humans. Thirty one cases of benign ovarian teratomas. (A total of 34 tumors including bilateral and multiple tumors) were examined. About one-third to half of the tumors showed LOH,which is caused by the reduction of either parental genome during meiotic division. In some cases, heterozygosity was maintained at one locus (IGF2) but was … More lost at another (SNRPN). This can be explained by the formation of crossing over of homologous chromatids. In benign teratomas, all three genes were expressed at very low levels, which is hardly detectable by Northern blotting. In contrast an immature teratoma. H19 was highly expressed, at levels almost equal to that of the third trimester placenta. IGF2 expression in an immature teratoma was low and similar to the level in dermoid cysts. Although the expression of the imprinted genes was low, cDNA could be amplified by RT-PCR from all samples of teratomas. In fourteen mature teratomas that were informative at the Apa I polymorphic site of IGF2, eight tumors (57%) showed biallelic expression. In H19, fourteen mature teratomas which were heterozygous either at the Rsa I or the Alu I polymorphic sites and biallelic expression was observed in 12 tumors (86%). In contrast, SNRPN expression was monoallelic in all the informative tumors (Table 1). In the tumors that showed monoallelic expression in either of the genes, the expressed allelotype was the same as that of the host. Examples of tumors showing biallelic (IGF2/H19) and monoallelic (SNRPN) expression were presented. The methylation status of H195'-promoter region was examined. The CpG sites within this region is known to be maternally hypomethylated. The adult somatic tissue was hemimethylated and sperm DNA was hypomethylated, which is compatible with previous reports. In ovarian teratomas, either mature (benign) or immature, this region is hypomethylated. Similarly, the methylation status of IGF2 exon 9 region, where the maternal allele is differentially hypomethylated, was analyzed. At this site, leukocyte DNA was hemimethylated, whereas sperm DNA was totally methylated. Ovarian teratomas had only hypomethylated alleles. For SNRPN,differentially methylated site is also demonstrated at its 5'-region, where the maternal allele is extensively methylated. The results of Southern blotting is presented. At this site,We have examined the allele specific expression of human imprinted genes in two cases of choriocarcinoma which had different genetic constitutions and seven choriocarcinoma cell lines. Case 1 and all four informative cell lines showed biallelic expression at the IGF2 site. Case also showed LOI for H19 but Case2 did not. Analyzes in cell lines revealed that 3/4 showed LOIat the H19 site. Our finding that the LOI of IGF2 and H19 is commonly associated with choriocarcinomas suggests its relevance to tumorgenesis. Less

已经发现有几个基因会受到基因组印记的影响。它们包括人类基因，如IGF2和Snrpn，它们只从父亲的等位基因表达，以及H19，它在大多数体细胞和胚胎外器官中从母体等位基因表达。为了检测印迹在卵巢畸胎瘤中的变异和鉴定印迹状态，我们研究了三个人类印迹基因(IGF2、H19和Snrpn)在一些皮样囊肿中的表达水平和等位基因特异性表达。我们还检查了等位基因的甲基化状态，其中印记特异性差异甲基化已在人类中得到证实。卵巢良性畸胎瘤31例。共检查34个肿瘤，包括双侧肿瘤和多发性肿瘤。大约三分之一到一半的肿瘤表现出杂合性缺失，这是由于减数分裂过程中亲本基因组的减少造成的。在某些情况下，杂合性保持在一个基因座(IGF2)，但为…更多的人在另一个地方迷路(SNRPN)。这可以用同源染色单体的交叉形成来解释。在良性畸胎瘤中，这三个基因的表达水平都很低，这一点在Northern blotting中很难检测到。相比之下，未成熟的畸胎瘤。H19高表达，几乎与妊娠晚期胎盘表达水平相当。IGF2在未成熟畸胎瘤中的表达较低，与皮样囊肿的表达水平相近。虽然印迹基因的表达水平较低，但RT-PCR均能从所有畸胎瘤标本中扩增出cDNAs。在14个IGF2基因Apa I多态位点有信息的成熟畸胎瘤中，8个肿瘤(57%)显示双等位基因表达。在H19中，14个成熟畸胎瘤在Rsa I或Alu I多态位杂合子，12个肿瘤中有双等位基因表达(86%)。相反，Snrpn在所有信息性肿瘤中的表达都是单等位基因(表1)。在这两个基因均呈单等位基因表达的肿瘤中，其表达的等位基因与宿主的等位基因相同。给出了表现出双等位基因(IGF2/H19)和单等位基因(Snrpn)表达的肿瘤的例子。检测H195‘-启动子区域甲基化状态。众所周知，该区域内的CpG位点是母系低甲基化的。成年体细胞组织半甲基化，精子DNA低甲基化，这与以前的报道一致。在卵巢畸胎瘤中，无论是成熟的(良性的)还是不成熟的，这一区域都是低甲基化的。类似地，分析了IGF2外显子9区域的甲基化状态，其中母亲等位基因的甲基化程度存在差异。在这个位置，白细胞DNA是半甲基化的，而精子DNA是完全甲基化的。卵巢畸胎瘤只有低甲基化等位基因。对于Snrpn，在其5‘-区也发现了差异甲基化位点，母体等位基因在那里被广泛甲基化。给出了Southern杂交的结果。在这一点上，我们已经检测了人类印记基因在两例不同基因构成的绒毛膜癌和七个绒毛膜癌细胞系中的等位基因特异性表达。病例1和所有四个信息性细胞系在IGF2位点显示双等位基因表达。病例还显示H19的LOI，但病例2没有。对细胞株的分析显示，3/4的细胞在H19位点有LOI。我们发现IGF2和H19的LOI通常与绒毛膜癌相关，这表明它与肿瘤的发生有关。较少

项目成果

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