胰腺导管腺癌中RASSF1肿瘤抑制基因：表达、染色体状态与表观遗传变化的相关性
RASSF1 tumor suppressor gene in pancreatic ductal adenocarcinoma: correlation of expression, chromosomal status and epigenetic changes.
作者信息
Amato Eliana, Barbi Stefano, Fassan Matteo, Luchini Claudio, Vicentini Caterina, Brunelli Matteo, Malleo Giuseppe, Scarpa Aldo, Malpeli Giorgio
机构信息
ARC-NET Centre for Applied Research on Cancer, Department of Pathology and Diagnostics, The Hospital and University of Verona, Verona, Italy.
Department of Pathology, The Hospital and University of Verona, Verona, Italy.
出版信息
BMC Cancer. 2016 Jan 12;16:11. doi: 10.1186/s12885-016-2048-0.
BACKGROUND
The Ras Association Domain Family Member 1 (RASSF1) is one of the most frequently reported methylation-inactivated tumor suppressor genes in primary pancreatic ductal adenocarcinomas (PDAC). Limited information is still available about the impact of RASSF1 gene silencing on the expression of its different isoforms in neoplastic cells.
METHODS
A series of 96 primary PDAC, with known clinico-pathological parameters, was tested for RASSF1 methylation status by methylation-specific PCR, RASSF1 locus copy number alterations by fluorescence in situ hybridization, and Rassf1a protein expression by immunohistochemistry. A further series of 14 xenografted primary PDAC and 8 PDAC-derived cell lines were tested to obtain a detailed methylation mapping of CpG islands A and C of the RASSF1 locus by pyrosequencing and to evaluate the expression of Rassf1 variants by qRT-PCR.
RESULTS
Methylation of CpG island A of the RASSF1 gene was observed in 35% of the tumors and allelic loss of RASSF1 locus was seen in 30 disomic and in 20 polysomic cases (52%). Rassf1a immunohistochemical expression was downregulated in half of primary PDAC, and this downregulation was neither correlated with methylation of RASSF1 promoter nor with RASSF1 copy number alterations. RASSF1 status did not influence patients' prognosis. The expression of the seven RASSF1 isoforms in xenografts and cell lines showed that RASSF1A, RASSF1B, and RASSF1C isoforms were present in all xenografts and cell lines, whereas RASSF1D, RASSF1E, and RASSF1F isoforms were variably expressed among samples. RASSF1G was never expressed in either xenografts or cell lines. The variable expression of RASSF1 isoforms in PDAC xenografts and cell lines was not dependent on RASSF1 methylation status of CpG islands A and C.
CONCLUSIONS
RASSF1 alterations occurring in PDAC mainly consist in variations of expression of the different isoforms. Different genetic mechanisms seem to contribute to RASSF1 deregulation in this setting, but RASSF1 methylation does not seem to substantially affect RASSF1 isoforms expression.
背景
Ras 关联结构域家族成员 1（RASSF1）是原发性胰腺导管腺癌（PDAC）中报道最为频繁的甲基化失活肿瘤抑制基因之一。关于 RASSF1 基因沉默对肿瘤细胞中其不同异构体表达的影响，目前仍知之甚少。
方法
对 96 例具有已知临床病理参数的原发性 PDAC 进行检测，通过甲基化特异性 PCR 检测 RASSF1 甲基化状态，通过荧光原位杂交检测 RASSF1 基因座拷贝数改变，通过免疫组织化学检测 Rassf1a 蛋白表达。另外对 14 例原发性 PDAC 异种移植瘤和 8 例 PDAC 衍生细胞系进行检测，通过焦磷酸测序获得 RASSF1 基因座 CpG 岛 A 和 C 的详细甲基化图谱，并通过 qRT-PCR 评估 Rassf1 变体的表达。
结果
在 35%的肿瘤中观察到 RASSF1 基因 CpG 岛 A 的甲基化，在 30 例二倍体和 20 例多倍体病例（52%）中观察到 RASSF1 基因座的等位基因缺失。在一半的原发性 PDAC 中，Rassf1a 的免疫组织化学表达下调，这种下调既与 RASSF1 启动子的甲基化无关，也与 RASSF1 拷贝数改变无关。RASSF1 状态不影响患者的预后。异种移植瘤和细胞系中七种 RASSF1 异构体的表达表明，RASSF1A、RASSF1B 和 RASSF1C 异构体存在于所有异种移植瘤和细胞系中，而 RASSF1D、RASSF1E 和 RASSF1F 异构体在样本中表达各异。RASSF1G 在异种移植瘤或细胞系中均未表达。PDAC 异种移植瘤和细胞系中 RASSF1 异构体的可变表达不依赖于 CpG 岛 A 和 C 的 RASSF1 甲基化状态。
结论
PDAC 中发生的 RASSF1 改变主要表现为不同异构体表达的变化。在这种情况下，不同的遗传机制似乎导致了 RASSF1 的失调，但 RASSF1 甲基化似乎并未实质性影响 RASSF1 异构体的表达。
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