Down-regulation of hedgehog-interacting protein through genetic and epigenetic alterations in human hepatocellular carcinoma
Down-regulation of hedgehog-interacting protein through genetic and epigenetic alterations in human hepatocellular carcinoma
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DOI：
10.1158/1078-0432.ccr-07-1181
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发表时间：
2008-06-15
影响因子：
11.5
通讯作者：
Omata, Masao
中科院分区：
文献类型：
--
作者：
Tada, Motohisa;Kanai, Fumihiko;Omata, Masao
Hedgehog（Hh）信号在几种癌症中被激活。然而，肝细胞癌（HCC）中Hh信号激活的机制尚未完全阐明。我们分析了HCC中Hh相互作用蛋白（HHIP）基因（Hh信号的负调节因子）的参与。胶质瘤相关癌基因同源物（Gli）报告基因测定，3-（4，5-二甲基噻唑-2-基）-5- 2-（3-羧基甲氧基苯基）（4-磺基苯基）-2H-四唑测定，在稳定表达HHIP的肝癌细胞中进行Hh信号的靶基因的实时定量逆转录- PCR。对肝癌细胞和36例肝癌组织进行HHIP实时定量PCR。采用亚硫酸氢钠测序、去甲基化分析和实时定量甲基化特异性PCR分析肝癌细胞和HCC组织的甲基化状态。结果：HHIP过表达诱导Gli报告基因活性、细胞活力和Hh信号靶基因转录水平显著降低。HHIP在肝癌细胞亚群中高甲基化并转录下调。用去甲基化剂处理导致HHIP DNA去甲基化和HHIP转录的恢复。HHIP转录在大多数HCC组织中也下调，超过一半的HCC组织中也下调。HHIP高甲基化。HHIP甲基化的HCC组织中HHIP转录水平显著低于HHIP未甲基化的HCC组织。结论：HHIP基因转录下调可能与DNA甲基化和/或洛有关，HHIP基因失活激活Hh信号可能参与了人肝癌的发病机制。
Purpose: Hedgehog (Hh) signaling is activated in several cancers. However, the mechanisms of Hh signaling activation in hepatocellular carcinoma (HCC) have not been fully elucidated. We analyzed the involvement of Hh-interacting protein (HHIP) gene, a negative regulator of Hh signaling, in HCC.Experimental Design: Glioma-associated oncogene homologue (Gli) reporter assay, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, and quantitative real-time reverse transcription - PCR for the target genes of the Hh signals were performed in HHIP stably expressing hepatoma cells. Quantitative real-time PCR for HHIP was performed in hepatoma cells and 36 HCC tissues. The methylation status of hepatoma cells and HCC tissues was also analyzed by sodium bisulfite sequencing, demethylation assay, and quantitative real-time methylation-specific PCR. Loss of heterozygosity (LOH) analysis was also performed in HCC tissues.Results: HHIP overexpression induced significant reductions of Gli reporter activity, cell viability, and transcription of the target genes of the Hh signals. HHIP was hypermethylated and transcriptionally down-regulated in a subset of hepatoma cells. Treatment with a demethylating agent led to the HHIP DNA demethylation and restoration of HHIP transcription. HHIP transcription was also down-regulated in the majority of HCC tissues, and more than half of HCC tissues. exhibited HHIP hypermethylation. The HHIP transcription level in HHIP-methylated HCC tissues was significantly lower than in HHIP-unmethylated HCC tissues. More than 30% of HCC tissues showed LOH at the HHIP locus.Conclusions: The down-regulation of HHIP transcription is due to DNA hypermethylation and/or LOH, and Hh signal activation through the inactivation of HHIP may be implicated in the pathogenesis of human HCC.