Epigenetic regulation identifies RASEF as a tumor-suppressor gene in uveal melanoma
Epigenetic regulation identifies RASEF as a tumor-suppressor gene in uveal melanoma
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DOI：
10.1167/iovs.07-1135
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发表时间：
2008-04-01
影响因子：
4.4
通讯作者：
van der Velden, Pieter A.
中科院分区：
文献类型：
--
作者：
Maat, Willem;Beiboer, Sigrid H. W.;van der Velden, Pieter A.
目的.最近，分离研究葡萄膜和皮肤黑色素瘤的家庭确定9 q21作为一个潜在的位点窝藏肿瘤抑制基因（TSG）。然后将该区域的一个基因RASEF作为候选TSG进行分析，但缺乏点突变和拷贝数变化无法证实这一点。本研究旨在探讨葡萄膜黑色素瘤中RASEF基因启动子甲基化引起的潜在突变和基因沉默。11葡萄膜黑色素瘤细胞系和35个原发性葡萄膜黑色素瘤样品中筛选RASEF基因突变的高分辨率熔解曲线和消化分析。RASEF的表达通过实时RT-PCR在所有细胞系和16个原发性葡萄膜黑色素瘤样品中测定，并通过直接测序分析和确认RASEF基因启动子的甲基化状态。突变筛查显示RASEF基因外显子5中存在一个已知的多态性（R262 C; C -> T），其频率正常（54%）。在原发性葡萄膜黑色素瘤中，46%呈现杂合基因型，11个细胞系中有10个（91%）呈现纯合基因型。熔解曲线分析表明，至少有两个原发性肿瘤的杂合性丢失。RASEF在细胞系和原发性肿瘤中的低表达与RASEF启动子区域的甲基化相关。原发肿瘤中RASEF基因的纯合性和甲基化与生存率降低相关（P = 0.019）。纯合性，结合甲基化，似乎是靶向RASEF葡萄膜黑色素瘤的机制，在这个位点的等位基因的不平衡支持RASEF的TSG的作用。
PURPOSE. Recently, a segregation study in families with uveal and cutaneous melanoma identified 9q21 as a potential locus harboring a tumor-suppressor gene (TSG). One of the genes in this area, RASEF, was then analyzed as a candidate TSG, but lack of point mutations and copy number changes could not confirm this. In this study, the RASEF gene was investigated for potential mutations and gene silencing by promoter methylation in uveal melanoma.METHODS. Eleven uveal melanoma cell lines and 35 primary uveal melanoma samples were screened for mutations in the RASEF gene by high-resolution melting-curve and digestion analysis. Expression of RASEF was determined by real-time RT-PCR in all cell lines and 16 primary uveal melanoma samples, and the methylation status of the promoter of the RASEF gene was analyzed and confirmed by direct sequencing.RESULTS. Mutation screening revealed a known polymorphism (R262C; C -> T) in exon 5 of the RASEF gene that displayed a normal frequency (54%). Of the primary uveal melanomas, 46% presented a heterozygous genotype, and 10 (91%) of 11 cell lines showed a homozygous genotype. Melting-curve analysis indicated loss of heterozygosity in at least two primary tumors. Low RASEF expression in the cell lines and primary tumors correlated with methylation of the RASEF promoter region. Homozygosity and methylation of the RASEF gene in primary tumors were associated with decreased survival (P = 0.019).CONCLUSIONS. Homozygosity, in combination with methylation, appears to be the mechanism targeting RASEF in uveal melanoma, and allelic imbalance at this locus supports a TSG role for RASEF.