Aberrant Promoter Methylation in Overexpression of CITED1 in Papillary Thyroid Cancer
Aberrant Promoter Methylation in Overexpression of CITED1 in Papillary Thyroid Cancer
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DOI：
10.1089/thy.2010.0295
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发表时间：
2011-05-01
期刊：
影响因子：
6.6
通讯作者：
Murata, Yoshiharu
中科院分区：
文献类型：
--
作者：
Sassa, Miho;Hayashi, Yoshitaka;Murata, Yoshiharu
背景：80%以上的甲状腺癌是最常见的内分泌恶性肿瘤，是甲状腺乳头状癌（PTC）。已经证实，CITED1 (Cbp/p300 interaction Transactivators with glutamic acid [E] and天冬氨酸[D]-rich c - end domain) mRNA在PTC中特征性过表达。我们之前的研究表明BRAF突变与CITED1过表达呈正相关。然而，CITED1在PTC中的表达机制尚不清楚。在本研究中，我们分析了CITED1基因的异常甲基化是否促进了PTC中CITED1的过表达。方法：采用定量聚合酶链反应分析19例PTC源性细胞系TPC1、K1、KTC-1以及手术切除的PTC及周围正常组织中CITED1 mRNA的表达水平。通过直接测序确定细胞和临床标本的BRAF突变状态。利用基因组DNA亚硫酸盐测序方法分析了CITED1启动子中脱氧柠檬酸-磷酸-脱氧鸟苷二核苷酸（CpGs）的甲基化状态。最后，分析了甲基化药物抑制TPC1细胞中cited1mrna的表达情况。结果：CITED1 mRNA在TPC1细胞中的表达水平低于K1和KTC-1细胞。BRAF突变存在于K1和KTC-1细胞中，但不存在于TPC1细胞中。在K-1和KTC-1细胞中，CITED1启动子被低甲基化，而在TPC1细胞中没有。在手术解剖标本中，PTC组织中cited1mrna的平均表达水平比正常组织高30倍。与PTC组织相比，正常组织中CITED1启动子中的CpGs甲基化程度更高。在没有BRAF突变的PTC标本中，两个CpGs的甲基化程度高于BRAF V600E突变的PTC标本。50-aza-20-脱氧胞苷对TPC1细胞甲基化的药理抑制导致cited1mrna表达增加。结论：CITED1启动子区CpGs的低甲基化与PTC组织中CITED1 mRNA的高表达相关，符合表观遗传调控参与CITED1过表达的假设。这一假设得到了TPC1细胞的药理学抑制研究的支持。
Background: More than 80% of all thyroid cancers, the most common endocrine malignancy, are papillary thyroid cancer (PTC). It is well established that CITED1 (Cbp/p300 Interacting Transactivators with glutamic acid [E] and aspartic acid [D]-rich C-terminal domain) mRNA is characteristically overexpressed in PTC. Our previous study suggested a positive association of BRAF mutation with CITED1 overexpression. However, the mechanism of CITED1 expression in PTC remains to be elucidated. In the present study, we analyzed whether aberrant methylation of CITED1 gene promotes CITED1 overexpression in PTC.Method: CITED1 mRNA expression levels were analyzed by quantitative polymerase chain reaction in three PTC-derived cell lines, TPC1, K1, and KTC-1, and in surgically dissected PTC and surrounding normal tissues from 19 patients. The BRAF mutation status of the cells and clinical specimens was determined by direct sequencing. The methylation status of the deoxycitidine-phosphate-deoxyguanosine dinucleotides (CpGs) in the CITED1 promoter was analyzed by the bisulfite-sequencing method using genomic DNA. Finally, the expression of CITED1 mRNA in TPC1 cells, when subjected to pharmacological inhibition of methylation, was analyzed.Results: CITED1 mRNA was expressed at lower levels in TPC1 than in K1 and KTC-1 cells. A BRAF mutation was present in K1 and KTC-1 cells, but not in TPC1 cells. CITED1 promoter was hypomethylated in K-1 and KTC-1 cells, but not in TPC1 cells. In surgically dissected specimens, the mean expression level of CITED1 mRNA was 30-fold higher in PTC tissues than in normal tissues. CpGs in the CITED1 promoter were more heavily methylated in normal tissues than in PTC tissues. In PTC specimens without a BRAF mutation, two CpGs were more heavily methylated than in PTC specimens with the BRAF V600E mutation. Pharmacological inhibition of methylation in TPC1 cells by 50-aza-20-deoxycitidine resulted in increased expression of CITED1 mRNA.Conclusion: Hypomethylation of the CpGs in the promoter region of CITED1 is associated with higher expression of CITED1 mRNA in PTC tissues, consistent with the hypothesis that epigenetic regulation is involved in the overexpression of CITED1. This hypothesis is supported by pharmacologic inhibition studies in TPC1 cells.