School of Public Health, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, China.
BMC Cancer. 2013 Dec 14;13:601. doi: 10.1186/1471-2407-13-601.
Glutaminase 2 (Gls2) is a p53 target gene and is known to play an important role in energy metabolism. Gls2 has been reported to be downregulated in human hepatocellular carcinomas (HCC). However, the underlying mechanism responsible for its downregulation is still unclear. Here, we investigated Gls2 expression and its promoter methylation status in human liver and colon cancers.
mRNA expression of Gls2 was determined in human liver and colon cancer cell lines and HCC tissues by real-time PCR and promoter methylation was analyzed by methylation-specific PCR (MSP) and validated by bisulfite genome sequencing (BGS). Cell growth was determined by colony formation assay and MTS assay. Statistical analysis was performed by Wilcoxon matched-pairs test or non-parametric t test.
First, we observed reduced Gls2 mRNA level in a selected group of liver and colon cancer cell lines and in the cancerous tissues from 20 HCC and 5 human colon cancer patients in comparison to their non-cancerous counter parts. Importantly, the lower level of Gls2 in cancer cells was closely correlated to its promoter hypermethylation; and chemical demethylation treatment with 5-aza-2'-deoxycytidine (Aza) increased Gls2 mRNA level in both liver and colon cancer cells, indicating that direct epigenetic silencing suppressed Gls2 expression by methylation. Next, we further examined this correlation in human HCC tissues, and 60% of primary liver tumor tissues had higher DNA methylation levels when compared with adjacent non-tumor tissues. Detailed methylation analysis of 23 CpG sites at a 300-bp promoter region by bisulfite genomic sequencing confirmed its methylation. Finally, we examined the biological function of Gls2 and found that restoring Gls2 expression in cancer cells significantly inhibited cancer cell growth and colony formation ability through induction of cell cycle arrest.
We provide evidence showing that epigenetic silencing of Gls2 via promoter hypermethylation is common in human liver and colon cancers and Gls2 appears to be a functional tumor suppressor involved in the liver and colon tumorigenesis.
谷氨酰胺酶 2（Gls2）是 p53 的靶基因，已知其在能量代谢中发挥重要作用。有报道称，Gls2 在人肝细胞癌（HCC）中下调。然而，其下调的潜在机制仍不清楚。在这里，我们研究了 Gls2 在人肝癌和结肠癌中的表达及其启动子甲基化状态。
通过实时 PCR 检测 Gls2 在人肝癌和结肠癌细胞系和 HCC 组织中的 mRNA 表达，通过甲基化特异性 PCR（MSP）分析启动子甲基化，并通过亚硫酸氢盐基因组测序（BGS）验证。通过集落形成实验和 MTS 实验测定细胞生长。通过 Wilcoxon 配对检验或非参数 t 检验进行统计学分析。
首先，我们观察到一组选定的肝癌和结肠癌细胞系以及 20 例 HCC 和 5 例人结肠癌患者的癌组织中 Gls2 的 mRNA 水平降低，与相应的非癌组织相比。重要的是，癌细胞中 Gls2 水平较低与启动子过度甲基化密切相关；用 5-氮杂-2'-脱氧胞苷（Aza）进行化学去甲基化处理可增加肝癌和结肠癌细胞中的 Gls2 mRNA 水平，表明直接的表观遗传沉默通过甲基化抑制 Gls2 表达。接下来，我们在人 HCC 组织中进一步研究了这种相关性，与相邻非肿瘤组织相比，60%的原发性肝肿瘤组织具有更高的 DNA 甲基化水平。通过亚硫酸氢盐基因组测序对 300bp 启动子区域的 23 个 CpG 位点进行详细的甲基化分析证实了其甲基化。最后，我们研究了 Gls2 的生物学功能，发现恢复癌细胞中的 Gls2 表达通过诱导细胞周期停滞显著抑制癌细胞生长和集落形成能力。
我们提供的证据表明，通过启动子过度甲基化的 Gls2 表观遗传沉默在人肝癌和结肠癌中很常见，并且 Gls2 似乎是一种功能性肿瘤抑制因子，参与肝癌和结肠癌的发生。
