Association Between The Activating Transcription Factor3(ATF3) Gene And Non-small Cell Lung Cancer

Background and Objective: Lung cancer has been the main cause of cancer deathworldwide and non-small cell lung cancer (NSCLC) cases accounts for nearly85%ofall the lung cancer cases. Up to86%of mortality associated with lung cancer is partiallyrelevant to the lack of early detection methods and a better understanding of evolutionof the disease and the molecular variation mechanisms is a priority. Activatingtranscription factor3(ATF3), a member of the ATF/cyclic AMP response elementbinding (ATF/CREB) family of transcription factors, has been implicated in thepathogenesis of several types of cancer. However, whether the expression of ATF3isaberrant in NSCLC and genetic variants or DNA methylation of the gene contribute tothe tumorigenesis of NSCLC are largely unknown. In the present study, the expressionof ATF3in four NSCLC cell lines was detected. Expression of ATF3in HumanBronchial Epithelial Cell (HBEpiC) used as a positive control.Then we explore thepromoter methylation status and mutation of the ATF3gene.Methods: Western blot was performed to determine the expression of ATF3genein four NSCLC cell lines (A549, NCI-H292,95C,95D) as well as HBEpiC. Quantity1.0software was carried out to estimate relative expression of ATF3protein for these celllines and β-Actin was calculated as an internal control for the semi-quantitative analysis.Bisulfite-sequencing PCR (BSP) to detect the promoter methylation status of the genein5cell lines and20NSCLC tissues and paired corresponding paracancerous lungtissues. The mutation of the5’-flanking1500-bp and coding sequence regions of theATF3gene were screened using DNA direct sequencing and single strand conformationpolymorphism (SSCP) respectively.Results: The expression of ATF3gene at protein level was significantly higher inNSCLC cell lines (A549, NCI-H292,95C,95D) than that in HBEpiC (P<0.01). MethylPrimer software analysis showed that in the ATF3gene promoter region sites-1175to -1039bp and-496to-288bp totally contain27CpG cites that are located in transcriptionfactor (TF) binding sites. There was completely unmethylated in these27CpG sites andno difference in the promoter regions of the ATF3gene between5detected cell lines,which were validated in lung cancer tissues and their corresponding paracarcinomatissues. No DNA mutation was found in ATF3coding sequence regions of60NSCLCtissues.Conclusion: The present study showed that the expression of ATF3protein wasincreased in NSCLC cells compared with that in normal bronchial epithelial cells,supporting the idea that up-regulated expression of ATF3contribute to thetumorigenesis of NSCLC. Genetic and epigenetic analyses revealed that the increasedATF3level was not attributed to aberrant methylation or variants in the promoter of thegene. In addition, our findings suggested that genetic variant in the CDS region of theATF3gene was not associated with NSCLC.