Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Dig Dis Sci. 2012 Oct;57(10):2608-14. doi: 10.1007/s10620-012-2202-8. Epub 2012 Jul 18.
Gene silencing via promoter hypermethylation plays a crucial role in the pathogenesis of cancers. Neuronal pentraxin II (NPTX2) has been observed to be hypermethylated in pancreatic cancers. Methylation of NPTX2 might provide a novel diagnostic marker for pancreatic cancers.
The objective of this study is to investigate the abnormal patterns of DNA methylation of NPTX2 in pancreatic cancers, and its role in the transcriptional silencing of NPTX2.
NPTX2 expression was detected by reverse-transcription polymerase chain reaction (RT-PCR), and the methylation status of NPTX2 was assessed by methylation-specific polymerase chain reaction (MSP). Immunohistochemistry was used to examine the NPTX2 protein expression. The pancreatic cancer cell lines were treated with the DNA methyltransferase inhibitor, histone deacetylase inhibitors, either alone or in combination.
The MSP analysis revealed that the promoter region of NPTX2 gene was largely unmethylated in normal pancreatic tissues, while NPTX2 was frequently hypermethylated in pancreatic cancer cells and in primary pancreatic carcinomas. Quantitative RT-PCR revealed that the mean mRNA expression level of NPTX2 in the pancreatic cancer tissues was significantly lower than that in the paired adjacent normal tissues (0.96 ± 0.91 vs. 2.78 ± 1.42, P < 0.001). Consistent with RT-PCR detection, treatment with 5Aza-dC resulted in different degrees of induction of NPTX2 protein in the various cancer cell lines.
We demonstrate that the NPTX2 protein is down-regulated in human primary pancreatic cancers and in pancreatic cancer cell lines. This study provides the first evidence that the down-regulation of NPTX2 tightly correlates with its promoter hypermethylation.
基因沉默通过启动子超甲基化在癌症的发病机制中起着至关重要的作用。神经元五联蛋白 II（NPTX2）已被观察到在胰腺癌中发生超甲基化。NPTX2 的甲基化可能为胰腺癌提供新的诊断标志物。
本研究旨在研究 NPTX2 在胰腺癌中的异常 DNA 甲基化模式及其在 NPTX2 转录沉默中的作用。
通过逆转录聚合酶链反应（RT-PCR）检测 NPTX2 的表达，通过甲基化特异性聚合酶链反应（MSP）评估 NPTX2 的甲基化状态。免疫组织化学用于检查 NPTX2 蛋白表达。用 DNA 甲基转移酶抑制剂、组蛋白去乙酰化酶抑制剂单独或联合处理胰腺癌细胞系。
MSP 分析显示，NPTX2 基因启动子区域在正常胰腺组织中基本非甲基化，而 NPTX2 在胰腺癌细胞和原发性胰腺癌中经常发生超甲基化。定量 RT-PCR 显示，胰腺癌组织中 NPTX2 的平均 mRNA 表达水平明显低于配对的相邻正常组织（0.96±0.91 对 2.78±1.42，P<0.001）。与 RT-PCR 检测一致，用 5Aza-dC 处理导致各种癌细胞系中 NPTX2 蛋白的不同程度诱导。
我们证明 NPTX2 蛋白在人原发性胰腺癌和胰腺癌细胞系中下调。本研究首次提供证据表明，NPTX2 的下调与启动子超甲基化密切相关。
