Chen Jung-Ta, Cheng Ya-Wen, Chou Ming-Chih, Sen-Lin Tong, Lai Wu-Wei, Ho William L, Lee Huei
Department of Pathology, Veteran General Hospital-Taichung, Taichung 402, Taiwan, ROC.
Clin Cancer Res. 2003 Sep 15;9(11):4200-4.
Reduced connexin (Cx) 43 gene expression has been shown in most of lung tumors and cancer cell lines. Although aberrant Cx43 gene expression was linked with lung tumorigenesis, our understanding to the mechanism was still limited. We hypothesized that the evidence of aberrant Cx43 gene expression was gradually intensified from adjacent normal lung tissues surrounding tumors toward tumor tissues. In this study, 90 lung tumors and adjacent normal tissues were collected to examine Cx43 mRNA expression by reverse transcription-PCR (RT-PCR). Our data showed that Cx43 mRNA expression in adjacent normal lung tissue was significantly correlated with nodal involvement (P = 0.03), but the similar trend was not observed in tumor tissues. To verify whether lack of Cx43 mRNA expression resulted from promoter methylation, PCR-based methylation assay was performed for Cx43 promoter methylation analysis. A higher frequency of promoter methylation was observed in Cx43 mRNA-negative patients (21 of 33, 63.7%) compared with Cx43 mRNA-positive patients (3 of 57, 5.3%, P < 0.0001). To elucidate whether aberrant Cx43 gene expression originated from adjacent normal lung tissues, 25 lung tumors and each of five adjacent normal tissues at various distances from tumor tissues were collected to examine Cx43 mRNA and protein expression by RT-PCR and Western blot, respectively. The results show that Cx43 mRNA and protein expressions gradually decreased from adjacent normal lung tissues to tumor tissues with a positive correlation to the distance from the tumor tissues. Gel-shift assay data also revealed that shifted band binding with AP1 was only observed in adjacent normal tissues, which were far from the tumor tissues. These results indicate that promoter methylation may interfere with AP1 binding to the promoter to cause aberrant Cx43 gene expression. Thus, Cx43 mRNA in adjacent normal tissue surrounding lung tumor simply detected by RT-PCR may act as a molecular marker of nodal micrometastasis in non-small cell lung cancer.
在大多数肺肿瘤和癌细胞系中，已发现连接蛋白（Cx）43基因表达降低。尽管Cx43基因表达异常与肺肿瘤发生有关，但我们对其机制的了解仍然有限。我们推测，从肿瘤周围的相邻正常肺组织到肿瘤组织，Cx43基因表达异常的证据会逐渐增强。在本研究中，收集了90例肺肿瘤及相邻正常组织，通过逆转录聚合酶链反应（RT-PCR）检测Cx43 mRNA表达。我们的数据显示，相邻正常肺组织中Cx43 mRNA表达与淋巴结受累显著相关（P = 0.03），但在肿瘤组织中未观察到类似趋势。为了验证Cx43 mRNA表达缺失是否由启动子甲基化引起，进行了基于PCR的甲基化检测以分析Cx43启动子甲基化情况。与Cx43 mRNA阳性患者（57例中的3例，5.3%，P < 0.0001）相比，在Cx43 mRNA阴性患者中观察到更高频率的启动子甲基化（33例中的21例，63.7%）。为了阐明Cx43基因表达异常是否起源于相邻正常肺组织，收集了25例肺肿瘤及距肿瘤组织不同距离的五个相邻正常组织中的每一个，分别通过RT-PCR和蛋白质印迹法检测Cx43 mRNA和蛋白质表达。结果显示，Cx43 mRNA和蛋白质表达从相邻正常肺组织到肿瘤组织逐渐降低，并与距肿瘤组织的距离呈正相关。凝胶迁移试验数据还显示，仅在远离肿瘤组织的相邻正常组织中观察到与AP1结合的迁移条带。这些结果表明，启动子甲基化可能干扰AP1与启动子的结合，从而导致Cx43基因表达异常。因此，通过RT-PCR简单检测肺肿瘤周围相邻正常组织中的Cx43 mRNA可能作为非小细胞肺癌淋巴结微转移的分子标志物。
