高甲基化下调 Runx3 基因表达，其恢复通过诱导人胃癌中 p27 和 caspase3 抑制胃上皮细胞生长。

Hypermethylation downregulates Runx3 gene expression and its restoration suppresses gastric epithelial cell growth by inducing p27 and caspase3 in human gastric cancer.

机构信息

Department of Gastroenterology, First Affiliated Hospital of Soochow University, China.

出版信息

J Gastroenterol Hepatol. 2010 Apr;25(4):823-31. doi: 10.1111/j.1440-1746.2009.06191.x.

DOI:10.1111/j.1440-1746.2009.06191.x

PMID:20492341

Abstract

BACKGROUND AND AIMS

Runx family transcription factors are integral components of transforming growth factor-beta signaling pathways and have been implicated in cell cycle regulation, differentiation, apoptosis, and malignant transformation. The silencing of tumor suppressor genes by aberrant hypermethylation occurs frequently in human cancer. It has been noted previously that Runx3 is regarded as an important tumor suppressor gene.

METHODS

Reverse transcription polymerase chain reaction was used to measure Runx3 and the DNA methyltransferase 1 (Dnmt1) messenger RNA (mRNA) expression level of paired samples of primary gastric cancer and corresponding non-cancerous gastric mucosae, which were obtained from surgically resected specimens of 70 patients. Western blot was used to detect the expression of Runx3 at protein levels. The promoter methylation status was measured by using methylation-specific polymerase chain reaction. We used Annexin V-FITC/PI assay to detect cell apoptosis, and the cell cycle was also analyzed. In order to examine the cell cycle and/or apoptosis, we determined p27 and caspase 3 expression by immunohistological analysis.

RESULTS

Our results demonstrate a loss or substantial decrease of Runx3 expression in 70 cases of gastric tumors as compared with that in normal gastric mucosa (0.5749 +/- 0.3580 vs 1.7252 +/- 0.4085, P < 0.05). The protein levels of the Runx3 gene were significantly lower in gastric cancers than those in adjacent normal tissues. The hypermethylation of Runx3 was involved in 50% (28/56) of gastric cancer tissues, which had reduced Runx3 mRNA expression. The differences of the Dnmt1 mRNA level were significant between the methylated and unmethylated Runx3 cancerous groups. Runx3 methylation was significantly correlated with increased Dnmt1 (r = 0.64, P < 0.01). Enforced restoration of Runx3 expression led to the induction of cell apoptosis and upregulation of p27 and caspase3 expression in vitro.

CONCLUSIONS

Our results suggest that a decrease of Runx3 expression by DNA hypermethylation is frequently associated with the evolution of gastric cancer. Runx3 was an independent prognostic factor and a potential therapeutic target for gastric cancer.

摘要

背景与目的

Runx 家族转录因子是转化生长因子-β信号通路的重要组成部分，参与细胞周期调控、分化、凋亡和恶性转化。肿瘤抑制基因的失活通过异常的高甲基化在人类癌症中经常发生。先前已经注意到，Runx3 被认为是一个重要的肿瘤抑制基因。

方法

使用逆转录聚合酶链反应测量 70 例原发性胃癌和相应非癌性胃黏膜的配对样本中 Runx3 和 DNA 甲基转移酶 1（Dnmt1）信使 RNA（mRNA）的表达水平，这些样本均来自手术切除的标本。使用 Western blot 检测 Runx3 蛋白水平的表达。通过甲基化特异性聚合酶链反应测量启动子甲基化状态。我们使用 Annexin V-FITC/PI 测定法检测细胞凋亡，还分析了细胞周期。为了检查细胞周期和/或细胞凋亡，我们通过免疫组织化学分析确定了 p27 和 caspase 3 的表达。

结果

与正常胃黏膜相比，我们的结果显示 70 例胃癌肿瘤中 Runx3 表达缺失或明显减少（0.5749 ± 0.3580 与 1.7252 ± 0.4085，P < 0.05）。胃癌组织中 Runx3 基因的蛋白水平明显低于相邻正常组织。Runx3 的高甲基化参与了 50%（28/56）具有低水平 Runx3 mRNA 表达的胃癌组织。甲基化和非甲基化 Runx3 癌组织之间的 Dnmt1 mRNA 水平差异显著。Runx3 甲基化与 Dnmt1 增加显著相关（r = 0.64，P < 0.01）。体外强制恢复 Runx3 表达导致细胞凋亡诱导和 p27 和 caspase3 表达上调。