10.14639/0392-100X-N1527
Objective. Laryngeal cancer is the second most common malignancy in the head and neck, with Epstein-Barr virus infection as a risk factor. Our aim is to evaluate correlations between the expression of lncRNA H19 and EBV infection in laryngeal cancer and H19 involvement in neoplastic progression through EZH2 association. Materials and methods. 30 paired laryngeal tissue specimens (neoplastic and non-neoplastic) were included in the study. Nucleic acid isolation and cDNA synthesis was performed according to the manufacturer’s protocol. EBV DNA and expression of lytic (BZLF1) and latent (LMP1) forms of infection were assessed in PCR assays; expression levels of H19 and EZH2 were quantified in qRT-PCR. Data was analysed using GraphPad Prism 5.0. Results. Higher H19 relative expression in neoplastic vs paired non-neoplastic samples was found (p &lt; 0.0001) with a significant increase in EBV DNA positive neoplasms (p = 0.0434). An inverse correlation between H19 and EZH2 expression levels was noticed in EBV positive cases. Additionally, increased levels of H19 in LMP1 positive samples compared with those positive for BZLF1 was found (p = 0.0593). Conclusions. lncRNA H19 and EZH2 significantly contribute to the development of laryngeal carcinoma, being correlated with EBV infection markers.
text/xml
1827-675X
1527
en
Gabriela Anton
http://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
infection marker
The association between lncRNA H19 and EZH2 expression in patients with EBV-positive laryngeal carcinoma
Text.Serial.Journal
https://www.actaitalica.it/
41
Laryngology
2021-11-26
2021-03-04
2021-12-22
2021-12-22
537-543
1
Gabriela Anton
2021/11/26
10.14639/0392-100X-N1527
537
Department of Molecular Virology, “Stefan S. Nicolau” Institute of Virology, Bucharest, Romania
1827-675X
infection marker
en
543
The association between lncRNA H19 and EZH2 expression in patients with EBV-positive laryngeal carcinoma
https://www.actaitalica.it/article/download/1527/704
41
ACTA OTORHINOLARYNGOLOGICA ITALICA 2021;41:537-543; doi: 10.14639/0392-100X-N1527




Laryngology


The association between lncRNA H19 and EZH2
expression in patients with EBV-positive laryngeal
carcinoma
L’associazione dell’espressione di lncRNA H19 e EZH2 nei pazienti affetti
da carcinoma laringeo EBV positivo
Irina E. Hotoboc1, Alina Fudulu1, Raluca Grigore2,3, Serban Bertesteanu2,3, Irina Huica1, Iulia V. Iancu1, Anca Botezatu1,
Coralia Bleotu1, Gabriela Anton1
1
  Department of Molecular Virology, “Stefan S. Nicolau” Institute of Virology, Bucharest, Romania; 2 “Carol Davila” University of
Medicine and Pharmacy, Bucharest, Romania; 3 ENT Department, Coltea Clinical Hospital, Bucharest, Romania


SUMMARY
Objective. Laryngeal cancer is the second most common malignancy in the head and neck,
with Epstein-Barr virus infection as a risk factor. Our aim is to evaluate correlations be-
tween the expression of lncRNA H19 and EBV infection in laryngeal cancer and H19 in-            Received: March 4, 2021
volvement in neoplastic progression through EZH2 association.                                   Accepted: May 3, 2021
Materials and methods. 30 paired laryngeal tissue specimens (neoplastic and non-neoplas-        Published online: November 26, 2021
tic) were included in the study. Nucleic acid isolation and cDNA synthesis was performed
according to the manufacturer’s protocol. EBV DNA and expression of lytic (BZLF1) and           Correspondence
latent (LMP1) forms of infection were assessed in PCR assays; expression levels of H19          Alina Fudulu
and EZH2 were quantified in qRT-PCR. Data was analysed using GraphPad Prism 5.0.                “Stefan S. Nicolau” Institute of Virology, De-
Results. Higher H19 relative expression in neoplastic vs paired non-neoplastic samples          partment of Molecular Virology, 285 Mihai Bravu
                                                                                                Ave, 030304 Bucharest, Romania
was found (p < 0.0001) with a significant increase in EBV DNA positive neoplasms
                                                                                                Tel./Fax +40 21 324.25.90
(p = 0.0434). An inverse correlation between H19 and EZH2 expression levels was noticed         E-mail: alina.fudulu@virology.ro
in EBV positive cases. Additionally, increased levels of H19 in LMP1 positive samples
compared with those positive for BZLF1 was found (p = 0.0593).                                  Funding
Conclusions. lncRNA H19 and EZH2 significantly contribute to the development of laryn-          The study was supported by Project 433/ID 929/
geal carcinoma, being correlated with EBV infection markers.                                    SMIS code14049.
KEY WORDS: lncRNA H19, EZH2, EBV, laryngeal carcinoma, infection marker
                                                                                                Authors contribution
                                                                                                All the authors have equal contribution.
RIASSUNTO
Obiettivo. Il cancro della laringe è il secondo tumore maligno più comune della testa e del     Conflict of interest
collo, l’infezione da virus Epstein-Barr (EBV) viene annoverata come fattore di rischio. Il     The Authors declare no conflict of interest.
nostro scopo è valutare le correlazioni tra l’espressione di lncRNA H19 e l’infezione da
EBV nel cancro della laringe e il coinvolgimento di H19 nella progressione della neoplasia        How to cite this article: Hotoboc IE, Fudulu A,
attraverso l’associazione di EZH2.                                                                Grigore R, et al. The association between lncR-
Materiali e metodi. Sono stati studiati 30 campioni di tessuto laringeo (neoplastici e non        NA H19 and EZH2 expression in patients with
neoplastici). Si è proceduto all’isolamento degli acidi nucleici e alla sintesi del cDNA se-      EBV-positive laryngeal carcinoma. Acta Otorhi-
condo specifico protocollo. Il DNA dell’EBV e l’espressione delle forme di infezione litica       nolaryngol Ital 2021;41:537-543. https://doi.
(BZLF1) e latente (LMP1) sono state valutate nei test con PCR; i livelli di espressione di        org/10.14639/0392-100X-N1527
H19 ed EZH2 sono stati quantificati mediante qRT-PCR. I dati sono stati analizzati utiliz-
zando GraphPad Prism 5.0.                                                                       © Società Italiana di Otorinolaringoiatria
                                                                                                e Chirurgia Cervico-Facciale
Risultati. È stata trovata una maggiore espressione relativa di H19 nei campioni neoplastici
rispetto a quelli non neoplastici (p < 0,0001) con un aumento significativo delle neoplasie
                                                                                                               OPEN ACCESS
positive al DNA di EBV (p = 0,0434). Una correlazione inversa tra i livelli di espressione di
H19 ed EZH2 è stata notata nei casi positivi per EBV. Inoltre, è stato riscontrato un aumento   This is an open access article distributed in accordance with
                                                                                                the CC-BY-NC-ND (Creative Commons Attribution-Non-
dei livelli di H19 nei campioni LMP1 positivi rispetto a quelli BZLF1 (p = 0,0593).             Commercial-NoDerivatives 4.0 International) license. The
Conclusioni. lncRNA H19 ed EZH2 contribuiscono in modo significativo allo sviluppo del          article can be used by giving appropriate credit and mentio-
carcinoma laringeo, essendo correlati con i marker di infezione da EBV.                         ning the license, but only for non-commercial purposes and
                                                                                                only in the original version. For further information: https://
PAROLE CHIAVE: lncRNA H19, EZH2, EBV, carcinoma laringeo, marker di infezione                   creativecommons.org/licenses/by-nc-nd/4.0/deed.en




                                                                                                                                                        537
I.E. Hotoboc et al.




Introduction                                                  Some reports have illustrated that non-coding RNAs may
                                                              be packed and secreted into circulating extracellular vesi-
Laryngeal carcinoma accounts for about 30-40% of              cles, thus playing a significant role in neoplasm growth,
head and neck malignancies 1. A multitude of risk fac-        metastatic processes and angiogenesis initiation, and drug
tors such as tobacco smoking, alcohol consumption, poor       resistance. Thus, lncRNAs expression may have diagnos-
oral hygiene, occupational and socioeconomic status,          tic and prognostic value, and many studies have investi-
dietary intake, hormonal factors and infectious agents        gated their potential as biomarkers 9. Specific lncRNAs
have been associated with the development of laryngeal        can lead to HNSCC (head and neck squamous cell carci-
carcinomas. In fact, smoking and alcohol consumption          noma) carcinogenesis, including laryngeal cancer. Among
have been shown to increase the risk of head and neck         these, H19 is suggested to be involved in the development
carcinoma, while genetic predisposition may play a role       of neoplastic cells and its overexpression in HNSCC has
in the initiation of laryngeal neoplasms 2. The synergis-     been associated with a low survival rate, a high risk of
tic actions create a local microenvironment favourable to     metastasis and disease recurrence 10. According to other
the development and progression of laryngeal carcinoma.       reports, miR-675, the mature product of this lncRNA, in-
Epidemiological reports have shown a close association        hibits the expression of pro-apoptotic genes by targeting
between viral infections and development of laryngeal         FADD (Fas-associated protein with death domain) in gas-
neoplasms. Some studies have highlighted the role of her-     tric cancer. EZH2 (Enhancer of zeste homolog 2) belongs
pesviruses in head and neck cancers, Epstein-Barr Virus       to mammalian epigenetic PRC2 complex that contributes
(EBV) and Kaposi Sarcoma Herpes Virus (KSHV) being            to trimethylation of H3K27, leading to chromatin silenc-
the best known oncogenic factors 3. Polz-Gruszka et al.       ing through its highly conserved SET domains in the C-
detected EBV in 57.5% of laryngeal and oropharyngeal          terminus. Acting as both a transcriptional suppressor and
carcinoma samples (laryngeal carcinomas accounting for        co-activator, EZH2 has an important role in cancer de-
60% of these samples), indicating an important role of        velopment and progression 11. The aim of this study was
this virus in the development and progression of this ma-     to evaluate the possible correlation between lncRNA H19
lignancy 2. Hasan et al. also detected (by in situ hybridi-   and neoplasm progression through EZH2 association in
zation - ISH) EBV small non-coding RNAs (EBERs) in            patients with EBV positive laryngeal cancer.
12/30 (40%) laryngeal carcinoma and 2/13 (15.4%) be-
nign laryngeal nodules, thus indicating viral contribution
to laryngeal malignant neoplasms 3. The EBV life cycle is     Materials and methods
divided into lytic and latent phases. The immediate-early
BZLF1 gene (BamHI Z fragment leftward open reading            Sample collection
frame 1) promotes EBV reactivation through its expressed      The study was performed on 60 paired tissue specimens
protein, Zta, and subsequently initiates the expression of    (neoplastic and adjacent non-neoplastic) obtained from 30
other EBV lytic genes in human epithelial cells. The tran-    patients (median age 59.5 years; range 44-83 years) diag-
sition between latent and lytic status might be linked to     nosed with laryngeal carcinoma, according to WHO crite-
the ability of BZLF1 to bind to methylated viral promot-      ria. Patient investigations and surgical interventions were
ers 4,5. The role of BZLF1 in lytic-latent switch has been    performed in the Department of Otorhinolaryngology at
associated with impaired DNA damage repair and abroga-        Coltea Clinical Hospital, Bucharest. The pathological stage
tion of the G2/M checkpoint, all leading to genomic in-       was determined according to TNM classification of the
stability in NPC (nasopharyngeal cancer) pathogenesis 6.      UICC. Clinical and epidemiological characteristics of the
Among latent genes of EBV, LMP1 (latent membrane              patients and samples are presented in Table I. The disease
protein 1) exhibits an anti-apoptotic and cellular prolif-    was significantly more frequent in men and smoking was a
erative role leading to the development of oral squamous      significant risk factor.
cell carcinoma 7. Some authors have suggested that LMP1       The pairs of laryngeal samples were obtained from patients
plays an important role in invasiveness of nasopharynge-      diagnosed with laryngeal carcinomas who underwent sur-
al epithelial cells in which its low levels may contribute    gery. The samples were preserved at -80°C immediately
to malignant transformation in the early stages of NPC        after harvesting and stored until nucleic acid isolation.
pathogenesis. For instance, Al-Thawadi et al. found EBV       All samples were collected with the patient’s informed
LMP1 expression in 14/25 laryngeal neoplasms 8. Over          consent and the study protocol was approved by the Ethic
the past decade, research has focused on the association      Committee of “St S. Nicolau” Institute of Virology (No
between lncRNAs (long non-coding RNAs) and cancers.           279/11.02.2019).



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                                                                                              H19 and EZH2 expression in EBV+ laryngeal carcinoma




Table I. The characteristics of study group.                                  mixture containing 5X PCR buffer (Promega GoTaq Green
 Gender                                   M                    28             Flexi Buffer), 25 mM MgCl2 (Promega), 10 mmol of each
                                          F                    2              dNTP (Promega), 10 pmol of each EBNA1 forward and re-
 Age                                    < 65                   15             verse primers, 5 U/μl Go-Taq DNA Polymerase (Promega)
                                     ≥ 65 years                15             and 200 ng of isolated DNA. PCR conditions were as fol-
 Smoking                                 Yes                   25             lows: 45 cycles consisting of 94°C for 1 minute, 58°C for
                                         No                     5             40 seconds, 72°C for 40 seconds followed by an extension
 T stage                               T1-T2                   14             of 7 minutes at 72°C. EBNA1 amplicons (138 bps) were
                                       T3-T4                   16             visualized in 2% agarose gel electrophoresis, after staining
 N stage                                 N0                     8             with ethidium bromide.
                                         N1                    14
                                                                              RNA isolation and cDNA synthesis
                                       N2-N3                    8
                                                                              Total RNA extraction from tissues was performed using Tri-
 M stage                                 M0                    26
                                                                              zol Reagent (InvitrogenTM, Thermo Fisher Scientific Inc.),
                                         M1                     4
                                                                              according to the manufacturer’s instructions. 50 mg of each
                                                                              tissue sample were disrupted using a homogeniser, and 1
                                                                              mL of Trizol reagent was added. The samples were further
DNA isolation                                                                 purified using RNeasy Mini kit (Qiagen, Hilden, Germany),
DNA isolation from tissues was performed using the High                       according to the manufacturer’s instructions and stored at
Pure PCR Template Preparation Kit (Roche Molecular Bio-                       -80°C until use. The purity and concentration of each RNA
chemicals, Mannheim, Germany), according to the manu-                         sample was determined using a NanoDrop ND1000 spectro-
facturer’s instructions. The DNA concentration and purity                     photometer (Thermo Fisher Scientific Inc.). The synthesis of
of each sample was determined using a NanoDrop ND1000                         cDNA was performed using Transcriptor First Strand cDNA
spectrophotometer (Thermo Fisher Scientific Inc.). All                        Synthesis Kit (Roche Molecular Biochemicals, Mannheim,
DNA samples were stored at -20°C.                                             Germany) according to the manufacturer’s instructions and
                                                                              each sample was stored at -20°C until use.
EBV DNA detection
EBV detection was performed by PCR using EBNA1 prim-                          BZLF1 and LMP1 PCR assay
ers (Tab. II). The reactions were performed in 25 μl volume                   Primers and TaqMan probes targeting BZLF1 (BamHI Z


Table II. Sequence of primers used in the study.
  Primer name                                                       Primer sequence                                         Reference
 EBNA1               Forward                                5’-TGATAACCATGGACGAGGAC-3’                                    Kahla et al., 12
                     Reverse                                 5’-CTTCAAGTTGCATTGGCTGC-3’
 BZLF1               Forward                            5’-AAATTTAAGAGATCCTCGTGTAAAACATC-3’                                Ryan et al., 13
                     Reverse                                5’-CGC CTC CTG TTG AAG CAG AT-3’
                      Probe                       5’-(6FAM)ATAATGGAGTCAACATCCAGGCTT GGGC(TAMRA)-3’
 LMP1                Forward                                  5’-CAGTCAGGCAAGCCTATGA-3’                                    Ryan et al., 13
                     Reverse                                  5’-CTGGTTCCGGTGGAGATGA-3’
                      Probe                           5’-(6FAM)GTCATAGTAGCTTAGCTGAAC(TAMRA)-3’
 H19                 Forward                                 5’-TGCTGCACTTTACAACCACTG-3’                               Mohammadi et al., 14
                     Reverse                                5’-ATGGTGTCTTTGATGTTGGGC-3’
 U6                  Forward                               5’-CTCGCTTCGGCAGCACATATACT-3’                                   Feng et al., 15
                     Reverse                                5’-ACGCTTCACGAATTTGCGTGTC-3’
 GAPDH               Forward                              5’-CCATCTTCCAGGAGCGAGATCCCT-3’                                  Iancu et al., 16
                     Reverse                               5’-TGAGCCCCAGCCTTCTTCATGGT-3’
 EZH2                Forward                              5’-TGCAACACCCAACACTTATAAGCGG-3’                                   This study
                     Reverse                               5’-CCTTTGCTCCCTCCAAATGCTGGT-3’




                                                                                                                                              539
I.E. Hotoboc et al.




fragment leftward open reading frame 1) and LMP1 (latent       N stage (N0/N1/N2&3) was noted and significant differ-
membrane protein-1) genes were used (Tab. II). PCR was         ences between non-neoplastic tissue samples and N0 stage
performed in 25 µL reaction volume (100 ng/ µL cDNA,           (p = 0.0080) as well as between N1 stage and N2&3 ones
1X TaqMan Universal MasterMix, 25 pmol of each forward         (p = 0.0253) were found (Fig. 1B).
and reverse primer, and 10 pmol TaqMan probe) using iQ5        The expression levels of EZH2 in laryngeal cancer were
System (BioRad, California, USA). Thermocycling condi-         evaluated (t-test) in neoplastic versus non-neoplastic
tions were: 50ºC for 2 minutes, 95ºC for 10 minutes, fol-      samples (Fig. 2A). Significant differences in EZH2 ex-
lowed by 40 cycles of 95ºC for 15 seconds and 60ºC for 1       pression (p = 0.0434) between neoplastic samples
minute. Specimens with Cq (quantification cycle) < 40 in       (mean = -1.361 ± 0.6765) and their non-neoplastic pair
the FAM channel were considered positive.                      (mean = -2.256 ± 1.120) were found. Next, potential asso-
                                                               ciation of EZH2 expression levels with clinicopathological
Quantitative real-time PCR analysis                            features was examined; t-test performed on these values re-
The expression levels of H19 and EZH2 were quantified          vealed increased expression for samples collected from pa-
in qRT-PCR on an iQ5 System (BioRad, California, USA)          tients with N0 stage, which was significant compared with
using Maxima SyBr Green/Fluorescein qPCR Master Mix            non-neoplastic (p = 0.0040) and N1 samples (p = 0.0010),
2x (Thermo Scientific Inc.) according to the manufacturer’s    and values similar with NT were found for N1 and N2/3
instructions. Specific primers were designed for targeted      samples (Fig. 2B).
genes using primer-BLAST (www.ncbi.nlm.nih.gov/tools/
primer-blast/). Primer sequences are presented in Table II.
The expression levels of H19 were normalised using the
U6 gene as reference while expression levels of EZH2 were
normalized using GAPDH gene. Each sample was tested
in duplicate. The qPCR data was analysed, and relative ex-
pression was calculated with Cq using 2−∆Cq method.

Statistical analysis
All statistical data was analysed using GraphPad Prism 5.0
(Graph Pad Software Inc., San Diego, CA, USA). The dif-
ferences between two groups were analysed using t-test
(Mann Whitney) and ANOVA (Kruskal-Wallis) for com-
paring three or more groups. Spearman non-parametric
correlation was performed with 95% CI. In all cases, a p       Figure 1. Relative expression levels of lncRNA H19 in neoplastic and adja-
value < 0.05 was considered statistically significant.         cent non-neoplastic laryngeal tissue samples (A); relative expression levels of
                                                               lncRNA H19 according to N stages (NT – non-neoplastic tissue samples) (B).
Results
The relative expression of lncRNA H19 in neoplastic
and adjacent non-neoplastic laryngeal tissues was in-
vestigated using qRT-PCR which revealed significantly
higher expression (p < 0.0001) in neoplastic samples
(mean = 1.574 ± 0.8629) vs paired non-neoplastic tissues
(mean = -0.4290 ± 0.8666) (Fig. 1A).
To investigate the clinical role of lncRNA H19 in laryngeal
cancer, potential correlations of its expression levels with
clinicopathological features were examined. Spearman
non-parametric correlation was performed, and a positive
association was noted between increases in lncRNA H19
expression with tumour size, although there was no sig-
nificant difference between consecutive groups (T1/T2/T3/
                                                               Figure 2. Relative expression levels of EZH2 in neoplastic vs adjacent non-
T4). No association with the metastasis status (M0 vs M1)      neoplastic laryngeal tissue sample (A); relative expression levels of EZH2 ac-
was observed. An increasing of H19 expression values with      cording to N stages (NT – non-neoplastic tissue samples) (B).



540
                                                                                             H19 and EZH2 expression in EBV+ laryngeal carcinoma




                                                                           Figure 4. The correlation between H19 and EZH2 expression levels in neo-
                                                                           plastic laryngeal samples (A) overall; (B) in EBV positive samples.




Figure 3. H19 expression levels in EBV-positive compared to EBV-negative
neoplastic tissue.


Viral testing confirmed the presence of the EBV genome
in 73.33% (22/30) of samples. The expression levels of
H19 in EBV DNA positive vs EBV DNA negative samples                        Figure 5. Relative expression of lncRNA H19 (A) and EZH2 (B) according to
was quantified. The results showed a significant increase of               EBV markers in laryngeal carcinoma.
the expression of this lncRNA in viral-positive neoplasms
(p = 0.0434) (Fig. 3).
Spearman’s correlation was used to investigate the relation                cesses associated with malignant diseases. Among these
between H19 and EZH2 expression levels in neoplastic sam-                  non-coding molecules, the potential of lncRNA H19 as a
ples (overall and EBV positive cases). The results indicated               biomarker has been evaluated in various types of cancers.
an inverse correlation between H19 and EZH2 expression                     Many authors indicated that lncRNA H19 is overexpressed
levels in EBV positive cases, highlighting a viral influence in            in cancer, thus suggesting its potential role in cell prolif-
the development of this type of carcinoma (Fig. 4).                        eration and tumorigenesis. lncRNA H19 overexpression
Markers of lytic and latent EBV infection were assessed by                 was found in pancreatic ductal adenocarcinoma, a neo-
TaqMan PCR assay and the results showed the expression                     plasm with an increased mortality, together with HOTAIR,
of BZLF1 in 31.82% and LMP1 in 18.18% of EBV posi-                         in plasma of patients with gastric cancer 17. In this study,
tive cases. Association between the expression of H19 and                  we found that lncRNA H19 was significantly upregulated
EZH2 and EBV markers in patients with laryngeal cancer                     in neoplastic versus normal tissue samples, and its expres-
was further evaluated (Fig. 5).                                            sion was associated with tumour size and N stage. We also
We found increased gene expression levels of lncRNA H19                    found significant differences between H19 expression lev-
in LMP1 positive samples compared with BZLF1positive                       els in N0 stage samples versus non-neoplastic tissues, re-
samples (p = 0.0593). In contrast, lower EZH2 gene ex-                     spectively, between the N1 stage and N2/N3 samples, but
pression levels were found in the LMP1 positive group ver-                 no significant association between lncRNA H19 and metas-
sus the BZLF1 positive group.                                              tasis stages (M0 and M1) was found. In contrast to these re-
                                                                           sults, some authors have reported a significant association
                                                                           of H19 levels with earlier lymph node metastasis, higher
Discussion                                                                 clinical stage, overall survival and disease-free survival 18.
In the last years, the clinical importance of long non-coding              Other meta-analysis revealed that lncRNA H19 expres-
RNAs has been recognised in different pathological pro-                    sion levels were closely associated with histological grade,



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I.E. Hotoboc et al.




TNM stage and degree of neoplasm invasion in gastrointes-        and our results consolidate the finding that lncRNA H19
tinal cancer and that it could be linked to distant metastasis   expression associates with latency and oncogenesis. This
in gastrointestinal and non-gastrointestinal cancers 19. We      EBV gene can be detected in exosomes isolated from the
found increased H19 expression in EBV-positive laryngeal         serum of NPC patients, thus facilitating its potential as a
neoplasms compared with EBV-negative ones, thus sug-             biomarker and underlying a clinical importance in NPC
gesting a possible key-role of EBV as a viral oncogenic          pathogenesis 24. On the other hand, Sun et al. found that
factor in initiation and progression of laryngeal neoplasms.     LMP1 could upregulate EZH2 in LMP1 positive HONE-1
EZH2 appears to play a dual role in cancer, either as an         NPC cell line, while other authors have indicated that
oncogene or tumour suppressor, so there are controversial        BZLF1 and BRLF1 promoters were correlated with a re-
reports regarding its expression levels in cancer. Increased     pressive hallmark of chromatin (H3K27me3), enhanced by
risk of metastasis and increased aggressiveness of malig-        an increased level of EZH2, in an EBV latent state 25.
nancy was found to be associated with loss of EZH2 in ad-
vanced stages 20.                                                Conclusions
In our study, we have shown that EZH2 expression levels
were increased in laryngeal neoplastic samples compared          The clinical importance of this research it related to its
to normal samples, thus suggesting the important func-           potential application in molecular diagnostics of this ma-
tion of this histone-methyltransferase in cell proliferation.    lignancy. This can be explained by the large variability of
However, the higher expression levels were found in sam-         the different diagnostic and prognostic biomarkers found in
ples collected from patients with N0 stage, suggesting a         many neoplasms, and thus the viral and cellular biomarkers
better correlation with early development of this type of        which play a specific role in LSCC development could of-
malignancy. In this study, we noticed a decrease in expres-      fer an interesting view of this pathology.
sion for patients with N+ stage at levels comparable with        We found increased lncRNA H19 expression in laryngeal
those found in non-neoplastic tissue samples. Other results      neoplasms associated with clinicopathological features (N
have indicated that EZH2 is overexpressed in laryngeal cell      stage). The highest values of H19 expression were noticed
carcinoma versus non-neoplastic tissues and that this cel-       in EBV-positive cases compared to EBV-negative ones
lular epigenetic marker could be considered as a poor prog-      with a close relationship between this non-coding molecule
nostic factor for nasopharyngeal and oral squamous cell          and EBV latent markers. On the other hand, an inverse cor-
carcinoma 21. Relative expression levels of EZH2 have also       relation between lncRNA H19 and EZH2 in laryngeal neo-
been associated with N stage in laryngeal carcinoma, em-         plasms was found, thus suggesting the role of this histone-
phasising its role in the progression of carcinogenesis. No      methyltransferase in cell proliferation. Further studies are
direct link between H19 and EZH2 has been reported, but          needed to elucidate the interplay between viral and epige-
rather through miR-630, an EZH2 repressor that interacts         netic factors in this pathology.
with H19 in a sequence-specific manner 22. In this study,        These future studies will be needed to better demonstrate
we found a negative correlation between H19 lncRNA and           the intimate relationship between viral markers (genes,
EZH2 expression levels with good values of Spearman cor-         non-coding products, proteins) and the cellular ones which
relation coefficient in neoplastic tissues samples, and even     define the entire variety of many signalling pathways from
better in EBV-positive neoplastic samples, in both cases         the following pathological processes (cellular proliferation,
with statistical significance. Furthermore, a statistically      neoplastic angiogenesis, cellular invasion and metastasis,
significant association between H19, EZH2 expression and         apoptosis, immune surveillance) involved in laryngeal can-
viral markers (BZLF1 and LMP1) presence could suggest            cer progression.
various reliable interpretations, depending on EBV infec-
tion patient’s status (latent infection, lytic reactivation).    References
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