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

Hotoboc, Irina Elisaveta; Fudulu, Alina; Huica, Irina; Iancu, Iulia Virginia; Grigore, Raluca; Bertesteanu, Șerban Vifor Gabriel; Bleotu, Coralia; Anton, Gabriela; Botezatu, Anca

doi:10.2478/rjim-2025-0005

Figures & Tables

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

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

Figure 3.

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

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

Sequences of primers and thermocycling conditions

Gene name [primers/probes reference]	Primer/Probe sequence	PCR 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′	qPCR 96ºC/10 min 40 cycles: 95ºC/10 s, 55ºC/30 s

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

Parameter	PDLIM4	WIF1	DAPK1
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)	*0.0186*	ns	*0.0259*

Clinical characteristics of patients based on EBV infection status

Clinical characteristics		EBV positive (n=11)	EBV negative (n=13)
Age (years)		57–77 (median: 66)	44–71 (median: 59)
Smoking	Yes	5 (20.8%)	13 (54.2%)
T stage	T1-T2	3 (12.5%)	9 (37.5%)
T stage	T3-T4	8 (33.3%)	4 (16.6%)
N stage	N0-N1	8 (33.3%)	3 (12.5%)
N stage	N2-N3	3 (12.5%)	10 (41.7%)
M stage	M0-M1	11 (45.8%)	13 (54.2%)

The characteristics of investigated group

Clinical characteristics		Number of cases [n (%)]
Age (years)	<65 / ≥65	11 (45.8%) / 13 (54.2%)
Smoking	Yes	19 (79.2%)
Lymph node metastasis	Yes / No	11 (45.8%) / 13 (54.2)
T stage	T1/T2	2/10 (8.3% / 41.7%)
T stage	T3/T4	9/3 (37.5% / 12.5%)
N stage	N0/N1	5/6 (20.8% / 25%)
N stage	N2/N3	12/1 (50% / 4.2%)
M stage	M0/M1	20/4 (83.3% / 16.7%)

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

Figures & Tables

Figure 1:

Figure 2.

Figure 3.

Figure 4.

Sequences of primers and thermocycling conditions

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

Clinical characteristics of patients based on EBV infection status

The characteristics of investigated group

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