Association Of Decorin Proximal Promoter And 5'-UTR Methylation Status With Non-small Cell Lung Cancer

Background and Objective: Lung cancer, including non-small cell lung cancer (NSCLC) and SCLC, is a leading cause of cancer death worldwide. DNA methylation has been considered to correlate with lung cancer, playing an important role in the development and progression of lung cancer. This study aims to investigate the association of Decorin gene proximal promoter region and 5'-UTR region methylation status with mRNA and protein expression in normal bronchial epithelial cell line and NSCLC cell lines, providing a theoretical basis for early diagnosis, prognosis and treatment of NSCLC.Methods: A PCR with universal primers and cloning sequencing were performed in three cell lines (Normal bronchial epithelial cell line HBE, Low metastatic giant-cell lung cancer cell line 95C, High metastatic giant-cell lung cancer cell line 95D) and three stage IV NSCLC tissues and paired corresponding paracancerous lung tissues to examine CpG sites methylation status of the proximal promoter region and 5'-UTR region of Decorin gene, then calculate the methylation frequencies of CpG sites located in the transcription factor (TF) binding sites. Meanwhile, fluorescent real-time quantitative PCR was carried out to analyze the mRNA level of Decorin gene in three cell lines. In addition, Enzyme-linked immunosorbent assay (ELISA) was applied to determine the Decorin protein level in three cell lines.Results: There are twenty CpG sites located in the proximal promoter region and 5'-UTR region of Decorin gene. According to Transcription Factor Binding Site Prediction software, we found that four CpG sites were located in the TF binding sites, which existed upstream from the transcription initial of Decorin, including -447bp, -391bp, -201bp and +58bp, and the corresponding TF were Oct-1, CDP CR, GATA-1, AhR-Ar individually. The sequencing results showed that the methylation frequencies of CpG sites located in CDP CR and AhR-Ar binding sites were 40%, 5%, 0% and 40%, 65%, 95% in three cell lines respectively. Statistical analysis showed that these two CpG sites methylation frequencies have significant difference among three cell lines (P<0.01). In addition, we detected these two CpG sites methylation frequencies in cancer and paired paracancerous lung tissues. The methylation frequencies of CpG sites which located in CDP CR binding sites were higher in paired paracancerous lung tissues than in cancer tissues, the situation of the methylation frequencies of CpG sites which located in AhR-Ar binding sites were opposite.The Real-time PCR results suggested the reduction of Decorin mRNA level was observed in three cell lines, and mRNA relative expression of Decorin gene had significant difference in three cell lines (P<0.05). The ELISA results showed that the reduction of Decorin protein expression in three cell lines were consistent with the mRNA expression of Decorin gene, and Decorin protein expression had significant difference in three cell lines (P<0.001).Conclusion: We discovered the methylation frequencies of CpG sites of Decorin gene proximal promoter region and 5'-UTR region, which CpG sites located in CDP CR and AhR-Ar binding sites, had significant difference in three cell lines. The fact that mRNA level expression of Decorin gene was reduced in three cell lines, it suggested the decrease of CpG methylation frequencies of CDP CR binding sites (CDP CR acts as a repressor in transcriptional regulation) and increase of CpG methylation frequencies of AhR-Ar bind sites (AhR-Ar acts as an activator in transcriptional regulation) may prevent the transcription of Decorin gene synergistically. Our results indicated that there was significant association between the methylation status of the proximal promoter region and 5'-UTR region of Decorin gene with its mRNA expression, providing new target for NSCLC clinical early diagnosis and new anti-tumor drug development.