Abstract
Highly methylated Long Interspersed Nucleotide Elements 1 (LINE-1) constitute approximately 20% of the human genome, thus serving as a surrogate marker of global genomic DNA methylation. To date, there is still lacking a consensus about the precise location in LINE-1 promoter and its methylation threshold value, making challenging the use of LINE-1 methylation as a diagnostic, prognostic markers in cancer. This study reports on a technical standardization of bisulfite-based DNA methylation analysis, which ensures the complete bisulfite conversion of repeated LINE-1 sequences, thus allowing accurate LINE-1 methylation value. In addition, the study also indicated the precise location in LINE-1 promoter of which significant variance in methylation level makes LINE-1 methylation as a potential diagnostic biomarker for lung cancer. A serial concentration of 5-50-500 ng of DNA from 275 formalin-fixed paraffin-embedded lung tissues were converted by bisulfite; methylation level of two local regions (at nucleotide position 300–368 as LINE-1.1 and 368–460 as LINE-1.2) in LINE-1 promoter was measured by real time PCR. The use of 5 ng of genomic DNA but no more allowed to detect LINE-1 hypomethylation in lung cancer tissue (14.34% versus 16.69% in non-cancerous lung diseases for LINE-1.1, p < 0.0001, and 30.28% versus 32.35% for LINE-1.2, p < 0.05). Our study thus highlighted the optimal and primordial concentration less than 5 ng of genomic DNA guarantees the complete LINE-1 bisulfite conversion, and significant variance in methylation level of the LINE-1 sequence position from 300 to 368 allowed to discriminate lung cancer from non-cancer samples.
Highlights
Alteration of CpG methylation status at either genome-wide or gene-specific level has been confirmed as powerful biomarkers for diagnosis, prognosis, and prediction of diseases [1]
As false positive result in DNA methylation analysis could be due to mismatches of primers to unspecific targets [31], the specificity of Long Interspersed Nucleotide Elements 1 (LINE-1) primers, designed to recognize
To monitor LINE-1 bisulfite conversion efficiency, either 5, 50 or 500 ng of genomic DNA extracted from 25 FFPE samples was mixed with either 106 or 108 copies (~ 0.4 ng) of Internal Control (IC) and submitted to bisulfite treatment
Summary
Alteration of CpG methylation status at either genome-wide or gene-specific level has been confirmed as powerful biomarkers for diagnosis, prognosis, and prediction of diseases [1]. Different commercial bisulfite conversion kits applied to various DNA sources have recently been comprehensively evaluated for their bisulfite conversion efficiency [7,8,9] In most of these performance evaluations, only one concentration of different target regions that are single copy sequences were chosen as reference limiting the in-depth performance assessment, if one wanted to analyze repeated sequences. Besides target concentrations, it has been reported that sequence complexity, GC content, secondary structure elements and even a given cytosine in a particular sequence can interfere with bisulfite conversion efficiency [6] In this delicate technical context, there are still lacking milestones guiding the determination of an optimized genomic DNA input that guarantees complete bisulfite conversion of repeated targets, which is critical to their correct methylation measurement. Not all CpG sites within a single promoter region are functionally equivalent in transcriptional regulation; the precise location of clinically relevant methylated CpGs plays an important role in the development of a DNA methylation-based biomarker [14, 15]
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