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The Association between DNA Methylation Status and Epstein-Barr Virus Infection in Laryngeal Carcinomas Cover

The Association between DNA Methylation Status and Epstein-Barr Virus Infection in Laryngeal Carcinomas

Open Access
|Mar 2025

Figures & Tables

Table 1

Sequences of primers and thermocycling conditions

Gene name [primers/probes reference]Primer/Probe sequencePCR technique Thermocycling conditions
β-globin [18]
  • Forward:5′-ACACAACTGTGTTCACTAGC-3′

  • Reverse:5′-CAACTTCATCCACGTTCACC-3′

  • PCR

  • 95°C/10 min

  • 40 cycles: 95°C/30 s, 55°C/50 s,

  • 72°C/1 min,

  • 72°C/7 min

BZLF1 [19]
  • Forward:5′-AAATTTAAGAGATCCTCGTGTAAAACATC-3′

  • Reverse:5′-CGCCTCCTGTTGAAGCAGAT-3′

  • Probe:5′-(6-FAM)-ATAATGGAGTCAACATCCAGGCTTGGGC-(TAMRA)-3′

  • qPCR

  • 50ºC/2 min, 95ºC/10 min

  • 40 cycles: 95ºC/15 s, 60ºC/1 min

LMP1 [19]
  • Forward:5′-CAGTCAGGCAAGCCTATGA-3′

  • Reverse:5′-CTGGTTCCGGTGGAGATGA-3′

  • Probe:5′-(6-FAM)-GTCATAGTAGCTTAGCTGAAC-(TAMRA)-3′

  • qPCR

  • 50ºC/2 min, 95ºC/10 min

  • 40 cycles: 95ºC/15 s, 60ºC/1 min

GAPDH [20]
  • Forward:5′-CCATCTTCCAGGAGCGAGATCCCT-3′

  • Reverse: 5′-TGAGCCCCAGCCTTCTTCATGGT-3′

  • qPCR

  • 96ºC/3 min

  • 40 cycles: 95ºC/10 s, 55ºC/30 s

PDLIM4 [21]
  • M-Forward:5′-TTTTAAAAGCGATTTTAAAGGGTC-3′

  • M-Reverse:5′-TTCGAAAAACTCTAAAAACCCG-3′

  • U-Forward:5′-TTTAAAAGTGATTTTAAAGGGTTGG-3′

  • U-Reverse:5′-CACTTCAAAAAACTCTAAAAACCCA-3′

  • qMS-PCR

  • 50ºC/2 min, 95ºC/10 min

  • 40 cycles: 95ºC/15 s, 58ºC/60 s

  • 50ºC/2 min, 95ºC/10 min

WIF1 [21]
  • M-Forward:5′-CGAGTTAAGGTTAGCGTTTGTTATC-3′

  • M-Reverse:5′-AAAATCCCTAATCAAAAAAAACATA-3′

  • U-Forward:5′-TTGAGTTAAGGTTAGTGTTTGTTATTG-3′

  • U-Reverse:5′-TAATAACTCCTATTCCTCCTCCAAC-3′

DAPK1 [this study]
  • M-Forward:5′-GGATAGTCGGATCGAGTTAACGTC-3′

  • M-Reverse:5′-CCCTCCCAACGCCGA-3′

  • U-Forward:5′-GGAGGATAGTTGGATTGAGTTAATGTT-3′

  • U-Reverse:5′-CAAATCCCTCCCAAACACCAA-3′

DNMT1 [20]
  • Forward:5′-GCGGTATACCCACCATGACA-3′

  • Reverse:5′-AGGCTTTGCCGGCTTCC-3′

  • qPCR

  • 96ºC/10 min

  • 40 cycles: 95ºC/10 s, 55ºC/30 s

DNMT3B [this study]
  • Forward:5′-CCAACAACACGCAACCAGTG-3′

  • Reverse:5′-CGTCTTCGAGTCTTGTTCTCGTA-3′

Table 2

The characteristics of investigated group

Clinical characteristicsNumber of cases [n (%)]
Age (years)<65 / ≥6511 (45.8%) / 13 (54.2%)
SmokingYes19 (79.2%)
Lymph node metastasisYes / No11 (45.8%) / 13 (54.2)
T stageT1/T22/10 (8.3% / 41.7%)
T3/T49/3 (37.5% / 12.5%)
N stageN0/N15/6 (20.8% / 25%)
N2/N312/1 (50% / 4.2%)
M stageM0/M120/4 (83.3% / 16.7%)
Figure 1:

The relationship between 5mC% and the expression levels of DNMTs in neoplastic tissues.

Figure 2.

Methylation percentages of PDLIM4, DAPK1 and WIF 1 genes’ promoters in neoplastic (T) and peri-neoplastic (PT) laryngeal tissues.

Table 3

The correlation between the percent of global (5mC%) and local (TSGs promoters) methylation

ParameterPDLIM4WIF1DAPK1
Pearson r−0.4203−0.2904−0.3996
95% CI−0.6743 to −0.07755−0.5763 to 0.05871−0.6604 to −0.05269
P value (two-tailed)0.0186ns0.0259
Table 4

Clinical characteristics of patients based on EBV infection status

Clinical characteristicsEBV positive (n=11)EBV negative (n=13)
Age (years)57–77 (median: 66)44–71 (median: 59)
SmokingYes5 (20.8%)13 (54.2%)
T stageT1-T23 (12.5%)9 (37.5%)
T3-T48 (33.3%)4 (16.6%)
N stageN0-N18 (33.3%)3 (12.5%)
N2-N33 (12.5%)10 (41.7%)
M stageM0-M111 (45.8%)13 (54.2%)
Figure 3.

Global (5-mC%) and local methylation profile (TSGs promoters methylation) in EBV-positive compared to EBV-negative laryngeal tumors.

Figure 4.

Investigated parameters in EBV positive (A) and EBV negative laryngeal tissue samples (B), according to T stage.

DOI: https://doi.org/10.2478/rjim-2025-0005 | Journal eISSN: 2501-062X (formerly 1220-4749) | Journal ISSN: 1220-4749
Language: English, Romanian
Page range: 152 - 162
Submitted on: Oct 16, 2024
Published on: Mar 21, 2025
Published by: N.G. Lupu Internal Medicine Foundation
In partnership with: Paradigm Publishing Services

© 2025 Irina Elisaveta Hotoboc, Alina Fudulu, Irina Huica, Iulia Virginia Iancu, Raluca Grigore, Șerban Vifor Gabriel Bertesteanu, Coralia Bleotu, Gabriela Anton, Anca Botezatu, published by N.G. Lupu Internal Medicine Foundation
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.