The role of methylation in regulation of the expression of the DAPK1 gene and associated microRNA genes in non-small cell lung cancer

Abstract

Background. Lung cancer is one of the most common malignant neoplasms. Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer, that accounts for more than 85% for all cases. The DAPK1 gene is one of the genes closely associated with the emergence and progression of this cancer. Epigenetic regulation of the DAPK1 gene occurs at different levels, in parti­ cular, by CpG island gene promoter methylation or by changes in the expression level of microRNAs, for which the DAPK1 gene is a target gene. The question of the effect of methylation and/or microRNAs on the regulation of the DAPK1 gene mRNA expression in NSCLC remains open. Aim. Detection of changes in the level of expression and/or methylation of microRNAs and their target gene DAPK1 in NSCLC. Methods. Samples of NSCLC tumors were collected and clinically characterized at the Research Institute of Clinical Oncology of the Blokhin National Research Center of Oncology. High-molecular DNA was isolated from the tissue by a standard method. The methylation level was determined using bisulfite DNA conversion and quantitative methyl-specific PCR (MS-PCR) with real-time detection. The levels of expression of 4 microRNAs and their putative target gene DAPK1 were determined by real-time PCR (RT-PCR). Statistical analysis was performed using an IBM SPSS 22 software package. The differences were considered significant at p<0.05. Results. The analysis with MS RT-PCR showed a statistically significant (p<0.05) increase in the level of methylation of the DAPK1 gene in tumor samples in comparison with paired histologically normal lung tissue. The level of the DAPK1 gene mRNA expression was statistically significantly associated with both the change in the methylation level of the DAPK1 gene promoter CpG island (Rs=-0.517, p=0.002) and the change in the expression of studied microRNA. The analysis of expression levels of DAPK1 and microRNAs allowed creation of two pairs, miR-339-3p – DAPK1 (Rs= -0.476, p=0.004) and miR-375 – DAPK1 (Rs= -0.354, p=0.037), which suggested a significant effect of these microRNAs on the regulation of DAPK1 gene activity. Conclusion. Thus, the newly discovered patterns are of interest for understanding the mechanisms of NSCLC development. They can form a basis for diagnosis and prognosis of this disease and also help adjustment of the treatment taking into account pathophysiological features of the tumor.