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Cancer screening is an important aspect of comprehensive health care, making a significant contribution to reducing the risk of death and the cost of treating patients. Finding new tumor markers with high sensitivity and specificity is a trend in the research activities in Vietnam as well as all over the world. Long interspersed element-1 (LINE-1), a large proportion of repeating DNA, is transposable to different positions in the genome. LINE-1 activity is controlled through DNA methylation (the CpG is attached with CH3), whereby LINE-1 is highly methylated in normal cells. However, in some types of cancer such as lung, breast, stomach, liver, rectum... changes in the methylation status LINE-1 have been noticed. To study the methylation status of the LINE-1 sequence, we used a quantitative methyl-specific PCR technique. This method requires a standard calibrator to quantify the rate of methylation. From the commercial methylated DNA (CpG Methylated Human Genomic DNA, Promega), we cloned two regions of the LINE-1 promoter corresponding to a reference sequence and an investigated one (target sequence) which are bisulfite transformed. As a result, we have created two recombinant plasmids, pRef-LINE1 and pMe-LINE1. The plasmids were mixed at 10% of methylation and could be used as a standard control for analyzing the DNA methylation of specimens from patients. Keywords: DNA methylation, DNA repeating sequence, LINE-1, quantitative methyl-specific-PCR (qMSP), tumor markers. References [1] A. P. Feinberg, Phenotypic Plasticity and the Epigenetics of Human Disease, Nature, Vol. 447, No. 7143, 2007, pp. 433-40, https://doi.org/10.1038/nature05919.[2] A. H. Ting, K. M. McGarvey, S. B. Baylin, The cancer Epigenome-Components and Functional Correlates, Genes Dev, Vol. 20, No. 23, 2006, pp. 3215-31, https://doi.org/10.1101/gad.1464906.[3] A. E. Teschendorff et al., DNA Methylation Outliers in Normal Breast Tissue Identify Field Defects that are Enriched in Cancer, Nat Commun, Vol. 7, No., 2016, pp. 10478, https://doi.org/10.1038/ncomms10478.[4] C. Leygo et al., DNA Methylation as a Noninvasive Epigenetic Biomarker for the Detection of Cancer, Dis Markers, Vol. 2017, No., 2017, pp. 3726595, https://doi.org/10.1155/2017/3726595.[5] X. Hao et al., DNA Methylation Markers for Diagnosis and Prognosis of Common Cancers, Proc Natl Acad Sci U S A, Vol. 114, No. 28, 2017, pp. 7414-7419, https://doi.org/10.1073/pnas.1703577114.[6] N. Kitkumthorn, A. Mutirangura, Long Interspersed Nuclear Element-1 Hypomethylation in Cancer: Biology and Clinical Applications, Clin Epigenetics, Vol. 2, No. 2, 2011, pp. 315-30, https://doi.org/10.1007/s13148-011-0032-8.[7] M. A. Kerachian, M. Kerachian, Long Interspersed Nucleotide Element-1 (LINE-1) Methylation in Colorectal Cancer, Clin Chim Acta, Vol. 488, No., 2019, pp. 209-214, https://doi.org/10.1016/j.cca.2018.11.018.[8] M. Barchitta et al., LINE-1 Hypermethylation in White Blood Cell DNA Is Associated with High-Grade Cervical Intraepithelial Neoplasia, BMC Cancer, Vol. 17, No. 1, 2017, pp. 601, https://doi.org/10.1186/s12885-017-3582-0.[9] Y. Baba et al., Long Interspersed Element-1 Methylation Level as a Prognostic Biomarker in Gastrointestinal Cancers, Digestion, Vol. 97, No. 1, 2018, pp. 26-30, https://doi.org/10.1159/000484104.[10] J. G. Herman et al., Methylation-specific PCR: a Novel PCR Assay for Methylation Status of Cpg Islands, Proc Natl Acad Sci U S A, Vol. 93, No. 18, 1996, pp. 9821-9826, https://doi.org/10.1073/pnas.93.18.9821.[11] K. Hattermann et al., A Methylation-Specific And SYBR-Green-Based Quantitative Polymerase Chain Reaction Technique for O6-Methylguanine DNA Methyltransferase Promoter Methylation Analysis, Anal Biochem, Vol. 377, No. 1, 2008, pp. 62-71, https://doi.org/10.1016/j.ab.2008.03.014. [12] K. J. Livak, T. D. Schmittgen, Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods, Vol. 25, No. 4, 2001, pp. 402-408, https://doi.org/10.1006/meth.2001.1262.[13] Y. Hou et al., Serious Overestimation In Quantitative PCR By Circular (Supercoiled) Plasmid Standard: Microalgal Pcna As The Model Gene, PLoS One, Vol. 5, No. 3, 2010, pp. e9545, https://doi.org/10.1371/journal.pone.0009545.[14] G. Johnson, T. Nolan, S. A. Bustin, Real-time Quantitative PCR, Pathogen Detection and MIQE, Methods Mol Biol, Vol. 943, 2013, pp. 1-16, https://doi.org/10.1007/978-1-60327-353-4_1.

Epigenetic alterations play a main role in the initiation and progression of lung cancer. CpG methylation in the promoter of the Short Stature Homeobox 2 (SHOX2) gene has been evaluated and validated at different stages of this malignant disease. Several approaches for measuring DNA methylation have been established, including quantitative methylation-specific PCR (qMSP). This is a simple, fast, and cost-effective technique that can be easily applied to clinical practice. In this study, formalin-fixed, paraffin-embedded (FFPE) tissue samples were collected from 30 lung cancer patients and 30 patients suffering from non-cancerous pulmonary diseases. The methylation level of SHOX2 was evaluated in two CpG-riched regions by using qMSP and one of them could be conferred as a potential biomarker to lung cancer.