The correlation between aberrant connexin 43 mRNA expression induced by promoter methylation and nodal micrometastasis in non-small cell lung cancer

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.