| Background and Objective: Lung cancer, divided into non-small cell lung cancer(NSCLC), accounting for~85%and small cell lung cancer (SCLC), accounting for~15%, is the leading cause of cancer-related death among various types of cancer in theworld. Owing to the scarcity of effective early diagnosis and therapy strategies, NSCLCis not only diagnosed at an advanced stage but also has a poor prognosis. Therefore,in-depth study of the molecular pathogenic mechanism is important for the preventionand treatment of NSCLC. TGF-β signaling plays important roles in the carcinogenesisand metastasis of the lung. Arkadia, encoded by gene RNF111, is a positive regulator ofTGF-β signaling through degradation of Smad7, SnoN and Ski. DNA methylation isconsidered to be the most typical epigenetic mechanism that correlates with lung cancerthrough regulating gene expression or silencing. In this study, we investigate the mRNAexpression of RNF111in NSCLC cell lines and human bronchial epithelial cell line. Inparticular, we aim to elucidate the underling mechanism of the mRNA expressiondifferences in NSCLC cell lines. Gene mutation? Gene promoter methylation? Theultimate goal is to provide an exciting new theoretical basis for early detection andtreatment of NSCLC.Methods: Fluorescent real-time quantitative PCR was performed to measure themRNA level of RNF111gene in cell lines, including human bronchial epithelial (HBE),low metastatic giant-cell lung cancer cell line (95C) and high metastatic giant-cell lungcancer cell line (95D). SSCP (Single Strand Conformation Polymorphism) was used todetect if mutation occurs at the promoter region or functional protein-coding region ofRNF111gene. Meanwhile, we analyzed the methylation status of CpG sites sited inRNF111promoter region in95C and95D cell lines through BSP and clone sequencing.Then we calculated the methylation frequencies of CpG sites of which methylationstatus are significantly different in two cell lines and vefified the relationship betweenthe different methylation frequencies and different mRNA level. In addition, EMSA (electrophoretic mobility shift assay) was carried out to verify whether these CpG sitesinfluence mRNA level via binding specific transcription factor.Results: The Real-time PCR results showed the mRNA relative expression ofRNF111gene was significantly different in95C and95D cell lines (P<0.01), suggestingthat the gene may be associated with aggressive tumor metastasis.No mutation was founded at the promoter region or functional protein-codingregion of RNF111gene in NSCLC tissues, paired paracancerous tissues and three celllines, indicating the different mRNA expression levels are not caused by mutations. TheBSP and sequencing results indicated that the methylation frequencies of-309,-109and+3CpG sites located in RNF111gene promoter was significantly different in95C and95D cell lines (P<0.01). The methylation frequencies of-309CpG site were0%and40%respectively in95C and95D cell lines, the-109CpG sites were45%and0%, the+3CpG sites were35%and5%. We predicted that the methylation status of CpG sitesdescribed above might regulate the gene transcription, leading to different levels ofmRNA expression in the two cell lines. The EMSA results demonstrated that the probesincluding-309,-109or+3CpG sites could combine with the nucleoprotein. However, itis only the probes including unmethylated and methylated-309CpG site could combinewith different amounts of nucleoprotein, suggesting that it might play important roles inthe regulation of gene transcription.Conclusion: In our study, we found the relative mRNA expression of RNF111genewas highest in95D and was definitely different from95C, indicating that RNF111genemight be related to lung cancer metastasis. It was proved that the different mRNA levelwas not caused by gene mutation. However, the methylation frequencies of-309,-109and+3CpG sites located in RNF111gene promoter was significantly different in95Cand95D cell lines. The EMSA results demonstrated that the probes including these CpGsites could combine with the nucleoprotein, but only the probes including unmethylatedand methylated-309CpG site could combine with different amounts of nucleoprotein,suggesting that it might play important roles in regulating gene transcription. Theprotein combining with the-309CpG site might be a negative regulation transcriptionfactor and its binding with DNA in95D cell line are prevented due to the increasedmethylation frequencies of-309CpG site, ultimatly resulting in high mRNA level. Next,we should clarify whether the protein is a transcription factor by MS. There wassignificant association between the methylation status of-309CpG site located in RNF111gene promoter and mRNA expression, providing new target for early diagnosisof NSCLC and anti-cancer drug development. |
