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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.

The three genes encoding small non‑coding microRNA (miR)34a, MIR34b and MIR34c act as tumor‑suppressor genes. Their aberrant expressions regulated by DNA methylation have been frequently found in various types of cancer. In the present study, the DNA promoter methylation profiles of the MIR34 gene family were analyzed using the methylation specific polymerase chain reaction in order to clarify their association with breast and lung cancer, non‑cancerous or normal adjacent tissues. The methylation frequency of MIR34a was significantly higher in breast cancer (49.37%) compared with normal adjacent tissues (30.38%). The methylation frequency of MIR34b/c was 59.49 and 62.03% in breast cancer and normal adjacent tissues, respectively. MIR34a methylation showed a significant concordance with that of MIR34b/c only in breast cancer tissue. MIR34a methylation was significantly associated with cancer and the invasive ductal carcinoma type of breast cancer (P=0.015 and P=0.02, respectively). Methylation frequency of MIR34a and MIR34b/c was 48.42 and 56.84% in lung cancer, and 47.22 and 51.39% in pulmonary diseases, respectively. No significant association was observed between the methylation status of MIR34a and MIR34b/c, and the clinicopathological features of lung cancer or with those of non‑cancerous pulmonary diseases. Promoter methylation of MIR34a and MIR34b/c occurs frequently and concomitantly in breast and lung cancer, as well as in pulmonary diseases tissues, but not in breast normal tissues adjacent to tumor. These results of the present study emphasize the involvement of MIR34 methylation in human diseases, including cancer. Furthermore, MIR34a methylation may be a promising marker for a subtype of breast cancer.

The significant differences in DNA methylation that are considered to be a biomarker for the diagnosis of cancer are a barrier to the application of biomarkers in the clinical field. In the present study, new primers were designed and further standard controls were set up to validate the accuracy of the methylation‑specific PCR (MSP), a method widely used to analyze DNA methylation. By analyzing the methylation pattern of breast cancer 1 (BRCA1) and estrogen receptor (ER) in 60 patients with breast cancer, the number of cases of methylated BRCA1 and ER detected by the primer was 7/60 and 21/60, respectively, whereas that detected by the previous widely used primers was 25/60 and 47/60, respec- tively. Sequencing of the MSP products indicated that the 18 and 26 false‑positive methylations of BRCA1 and ER, respec- tively, were due to insufficient validation of the previously used primers. Thus, the present study proposes that all studies based on the MSP approach should incorporate more controls to validate the precision of the MSP primers.

DNA ladder is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare a DNA ladder that consists of 10 fragments from 100 to 1000 bp. This protocol is a combination of routinely employed methods: cloning, PCR, and partial digestion with restriction enzymes. DNA fragments of 100 bp with unique restriction site at both ends were self-ligated to create a tandem repeat. Once being cloned, the tandem repeat was rapidly amplified by PCR and partially digested by restriction enzymes to produce a ladder containing multimers of the repeated DNA fragments. Our procedure for production of DNA ladder could be simple, time saving, and inexpensive in comparison with current ones widely used in most laboratories.