Detection of the hypennethylation of MLH1 promoter and its clinical application in endometrial cancer screening

MLH1启动子高甲基化检测及其在子宫内膜癌筛查中的临床应用

• 批准号: 13557137
• 负责人: INOUE Masaki
• 金额: $ 7.1万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13557137/
• 关键词: DNA mismatched repair genes; human MLH1; hypermethylation of promoter sequences; endometrial cancer; molecular targets; cancer screening program; テロメラーゼ; エピジェネティック変化; DNAメチル化; MLH1遺伝子; 子宮内膜癌; hMLH1遺伝子; プロモーターのメチル化; 遺伝子診療; PTEN遺伝子; 癌リスク因子

Silencing of the MLH1 gene by promoter hypermethylation is the main mechanism underlying the microsatellite instability(MSI) phenotype in endometrial cancers. MSI has a key role in the endometrial carcinogesis where mutations of multiple genes have involved.We have developed the convenient and sensitive method for the detection of promoter hypermethylation in the region 700bp upstream of MLH1 covering 48 CpG sites. The metylation of these sites has been confirmed by bisulfate sequencing. Metylation status was classified as full(over 80% of CpGs are methylated), partial(10-80%) or nonmethylation(less than 10%). Of endometrial cancers examined, 30% were fully methylated, 25% were partially methylated and 45% were not methylated. Analysis of MLH1 by immunohistochemical methods and of MSI revealed that the degree, rather than region-specific methylation of CpG island is critical for decreased MLH1 expression and the MSI phenotype. Among patients with methylated cancers, almost half patients have contained methylated promoters in their normal endometria with profiles similar to those of cancerous lesions, and these were closely associated with the MSI phenotype. In contrast, only a few cases of normal endometria from patients without endometrial malignancies harbored methylated promoters. The present study suggests that hypermetylation of the MLH1 promoter is frequent in the histologically-cofirmed normal endometrium adjacent to cancerous lesions, supporting the notion that hypermethylation of DNA-mismatch repair genes is the initial step that triggers the following various genetic events in the endometrial carcinogenesis. Of course, the genetic events could be candidates for molecular targets in the diagnosis and treatment.Detection of some molecular targets in a tiny clinical sample might be a useful diagnostic aid in cancer screening.

通过启动子高甲基化对MLH1基因的沉默是子宫内膜癌中微卫星不稳定性（MSI）表型的主要机制。 MSI在涉及多个基因的突变的子宫内膜致癌中具有关键作用。我们开发了一种方便而敏感的方法，用于检测MLH1上游的启动子高甲基化的启动子高甲基化，覆盖48 CpG位点。这些位点的基因化已通过硫酸盐测序证实。将基因化状态分类为完全（超过80％的CpG是甲基化的），部分（10-80％）或非甲基化（小于10％）。在检查的子宫内膜癌中，有30％被完全甲基化，25％被部分甲基化，而45％未甲基化。通过免疫组织化学方法和MSI对MLH1的分析表明，CPG岛的程度而不是区域特异性甲基化对于降低MLH1表达和MSI表型至关重要。在甲基化癌症的患者中，几乎一半的患者在正常的内部测量室中含有甲基化的启动子，其特征与癌变病变相似，并且与MSI表型密切相关。相反，没有子宫内膜恶性肿瘤的患者只有少数正常的内腹病例置换了甲基化的启动子。本研究表明，在与癌变病变相邻的组织学确认的正常子宫内膜中，MLH1启动子的高型甲基化频繁，这支持了以下观点：DNA匹配修复基因的高甲基化是子宫内膜致癌物中以下各种遗传事件的初始步骤。当然，遗传事件可能是诊断和治疗中分子靶标的候选者。在微小的临床样本中进行某些分子靶标可能是对癌症筛查的有用诊断帮助。

项目成果

Tanaka M, Kyo S, Inoue M et al.: "Evidence of monoclonal composition of human endometrial glands and masaic pattern of clonal distribution"Am J Pathol. 163. 295-301 (2003)

Tanaka M、Kyo S、Inoue M 等人：“人类子宫内膜腺体单克隆组成和克隆分布马赛克模式的证据”Am J Pathol。

Kyo S, Inoue M et al.: "Significance of immunological detection of hTERT"Am J Pathol. 163. 859-869 (2003)

Kyo S、Inoue M 等人：“hTERT 免疫学检测的意义”Am J Pathol。

Kyo S, Masutomi K, Maida M, Kanaya T, Yatabe N, Nakamura M, Takakura M, Suga\yara I, Murakami S, Taira T, Inoue M.: "Successful immortalization of endometrial glandular cells with normal structural and functional characteristics."Am J Pathol. 163. 2259-22

Kyo S、Masutomi K、Maida M、Kanaya T、Yatabe N、Nakamura M、Takakura M、Sugayara I、Murakami S、Taira T、Inoue M.：“具有正常结构和功能特征的子宫内膜腺细胞成功永生化。

Sasagawa T, Inoue M et al.: "Mucosal immunoglobulin-A and -G responses to oncogenic HPV capsids"Int J Cancer. 104(3). 328-335 (2003)

Sasakawa T、Inoue M 等人：“粘膜免疫球蛋白-A 和 -G 对致癌 HPV 衣壳的反应”Int J Cancer。

Kyo S, Kanaya T, Inoue M.: "Role of hMLH1 gene hypermethylation in endometrial carcinogenesis."Cell and Molecular Biology of Endometrial Carcinoma, (kuramoto H, Nishida M(Eds.)(springer-Verlag, New York). 232-244 (2003)

Kyo S、Kanaya T、Inoue M.：“hMLH1 基因高甲基化在子宫内膜癌发生中的作用。”子宫内膜癌的细胞和分子生物学，（kuramoto H、Nishida M（编辑）（springer-verlag，纽约）。232-