Dynamics of LINE-1 Retrotransposon Methylation Levels in Circulating DNA from Lung Cancer Patients Undergoing Antitumor Therapy

Abstract

Malignant cell transformation is accompanied with abnormal DNA methylation, such as the hypermethylation of certain gene promoters and hypomethylation of retrotransposons. In particular, the hypomethylation of the human-specific family of LINE-1 retrotransposons was observed in lung cancer tissues. It is also known that the circulating DNA (cirDNA) of blood plasma and cell-surface-bound circulating DNA (csb-cirDNA) of cancer patients accumulate tumor-specific aberrantly methylated DNA fragments, which are currently considered to be valuable cancer markers. This work compares LINE-1 retrotransposon methylation patterns in cirDNA of 16 lung cancer patients before and after treatment. CirDNA was isolated from blood plasma, and csb-cirDNA fractions were obtained by successive elution with EDTA-containing phosphate buffered saline and trypsin. Concentrations of methylated LINE-1 region 1 copies (LINE-1-met) were assayed by real-time methylation-specific PCR. LINE-1 methylation levels were normalized to the concentration of LINE-1 region 2, which was independent of the methylation status (LINE-1-Ind). The concentrations of LINE-1-met and LINE-1-Ind in csb-cirDNA of lung cancer patients exhibited correlations before treatment (r = 0.54), after chemotherapy (r = 0.72), and after surgery (r = 0.83) (P < 0.05, Spearman rank test). In the total group of patients, the level of LINE-1 methylation (determined as the LINE-1-met/LINE-1-Ind ratio) was shown to increase significantly during the follow-up after chemotherapy (P < 0.05, paired t test) and after surgery compared to the level of methylation before treatment (P < 0.05, paired t test). The revealed association between the level of LINE-1 methylation and the effect of antitumor therapy was more pronounced in squamous cell lung cancer than in adenocarcinoma (P < 0.05 and P > 0.05, respectively). These results suggest a need for the further investigation of dynamic changes in levels of LINE-1 methylation depending on the antitumor therapy.