RNA sequencing of head and neck squamous cell carcinoma (HNSCC) patients who drank alcohol or did not identified two long non-coding RNAs (lncRNAs), lnc-PSD4-1 and lnc-NETO-1, that were differentially expressed due to alcohol consumption. Normal oral keratinocytes exposed to ethanol and acetaldehyde showed dysregulation of these lncRNAs, validating the RNA-seq results. Low expression of lnc-PSD4-1:14 strongly correlated with higher survival rates in HNSCC patients.

1. RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
Alcohol consumption has been
implicated in the pathogenesis of head
and neck squamous cell carcinoma
(HNSCC), though its mechanism is
poorly understood. Long non-coding
RNA (lncRNA) is a noncoding RNA that
has a profound effect on numerous
biological processes including
tumorigenesis and oncogenesis. Our
central hypothesis is that alcohol-
mediated dysregulation of lncRNAs is a
key event in HNSCC pathogenesis.
Utilizing TCGA RNA-seq data from 34
HNSCC patients which included alcohol
drinkers and non-drinkers, we analyzed
the expression levels of lncRNAs to
identify a set of differentially expressed
lncRNAs due to alcohol consumption.
Normal oral keratinocytes were
exposed to ethanol and acetaldehyde
to validate the RNA sequencing results
we obtained. A long-term survival
analysis was then conducted on the
verified lncRNAs utilizing a Cox
Proportional-Hazards regression model
and Kaplan-Meier survival curve. We
identified two lncRNAs: lnc-PSD4-1
and the various splice variants of lnc-
NETO-1 that were differentially
expressed due to alcohol consumption
from RNA-seq analysis of the clinical
data. The oral keratinocytes exposed to
alcohol and acetaldehyde also
demonstrated dysregulation of these
two lncRNAs, validating the RNA-seq
analysis. In addition, low expression of
lnc-PSD4-1:14 showed a strong
correlation with higher survival rates.
lnc-PSD4-1:14 and lnc-NETO-1
potentially play a key role in the early
pathogenesis of HNSCC since they are
dysregulated in both clinical data and
in vitro experiments mimicking the
effects of alcohol use.
Abstract
RNA-seq Analysis
RNA-seq libraries
composed of 34 HNSCC
patients, 17 drinkers and
17 nondrinkers were
obtained from The
Cancer Genome Atlas.
The BEDtools utility
coverageBed was utilized
to generate lncRNA read
counts (integer values of
expression level). These
read counts were then
put through a lncRNA
differential expression
analysis comparing
drinkers versus
nondrinkers.
Materials and Methods
Differentially Expressed lncRNAs
RNA-Seq differential expression analysis
between drinkers and nondrinkers revealed
11 statistically significant lncRNAs.
In Vitro Verification
Of the 11 lncRNAs identified in the RNA-seq
analysis, two were verified: PSD4-1 (including
PSD4-1:14) and NETO1-1, with both having
increased expression levels in the ethanol and
acetaldehyde treated samples compared to
the non-treated controls.
Long-Term Survival Analysis
Low expressions of PSD4-1:14 were highly
correlated with overall better patient survival
in both univariate and multivariate models.
Results Conclusions
The increased expression of NETO1-1
and PSD4-1:14 in both HNSCC patient
clinical samples and in vitro models of
alcohol usage suggest that NETO1-1
and PSD4-1:14 may act as promoters
of oncogenes. While further study is
necessary to understand the molecular
mechanisms by which these lncRNAs
work, PSD4-1 and NETO1-1 hold
promise as potential biomarkers, and
therapeutic targets of HSNCC, that
could lead to improvement in the
treatment of this disease.
Figure Legend
1) Deng G, Sui G (2013) Noncoding RNA in oncogenesis: a new era of
identifying key players. Int J Mol Sci 14: 18319-18349.
2) Homann N, Jousimies-Somer H, Jokelainen K, Heine R, Salaspuro M
(1997) High acetaldehyde levels in saliva after ethanol consumption:
methodological aspects and pathogenetic implications. Carcinogenesis
18: 1739-1743.
3) Hashibe M, Brennan P, Benhamou S, Castellsague X, Chen C, et al.
(2007) Alcohol drinking in never users of tobacco, cigarette smoking in
never drinkers, and the risk of head and neck cancer: pooled analysis
in the International Head and Neck Cancer Epidemiology Consortium.
J Natl Cancer Inst 99: 777-789.
Acknowledgments
• Calit2 and Qualcomm Institute Summer Scholar Program
• Dr. Jessica Wang-Rodriguez
1Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of California San Diego
2 Veterans Administration Medical Center and Department of Pathology, University of California San Diego
Pranav Singh1 Vicky Yu1, Elham Rahimy1, Hao Zheng1, Selena Z. Kuo1, Elizabeth Kim1, Weg M. Ongkeko1.
Jessica Wang-Rodriguez2
RNA-seq analysis identifies key long non-coding RNAs connected to the
pathogenesis of alcohol-associated head and neck squamous cell carcinoma
Log(2)FC Log(2)CPM p-value FDR
lnc-SLC39A11-2:7 4.194151 3.878865 1.94E-07 0.001085
lnc-SLC39A11-2:5 4.140367 3.885084 2.44E-07 0.001085
lnc-SLC39A11-2:6 4.142305 3.884648 2.46E-07 0.001085
lnc-LAMB3-1:1 -2.581367 6.606811 2.54E-06 0.008414
lnc-CCL18-1:1 -2.190396 5.522936 5.69E-06 0.015064
lnc-NETO1-1:9 3.351077 3.585717 1.56E-05 0.022272
lnc-NETO1-1:3 3.364047 3.615927 1.63E-05 0.022272
lnc-NETO1-1:2 3.362404 3.616193 1.64E-05 0.022272
lnc-NETO1-1:4 3.347929 3.625721 1.68E-05 0.022272
lnc-NETO1-1:6 3.34735 3.624247 1.68E-05 0.022272
lnc-PSD4-1:14 1.669653 1.020086 2.74E-05 0.032967
lnc-SPANXA2-2:1 2.367287 2.927082 3.36E-05 0.037051
lnc-AC002472.13.1-1:1 -1.862271 1.547773 4.26E-05 0.041177
lnc-ERC1-1:2 -3.066857 4.256589 4.98E-05 0.041177
lnc-FBXL14-1:1 -3.066857 4.256589 4.98E-05 0.041177
lnc-ERC1-1:3 -3.066857 4.256589 4.98E-05 0.041177
lnc-KTN1-AS1-1:6 -2.463648 1.922281 5.81E-05 0.042703
Based upon
low expression
Univariate
Hazard Ratio
(95% CI)
p-value Multivariate
Hazard Ratio
(95% CI)
p-value
LncRNA PSD4-
1:14
0.267150
( 0.072234-
0.988021)
0.047926 0.236208
(0.062212-
0.896836)
0.034013
Ethanol/Acetaldehyde Treatments
We treated the normal oral keratinocytes
(OKF4, OKF6) with increasing dosages of 200
proof ethanol: 0%, 0.1%, and 0.3% ethanol by
volume. To represent long-term alcohol use,
we treated the cells every 24 hours with
ethanol diluted in medium for a period of 28
days. We also repeated treatment with
acetaldehyde, the first metabolite of ingested
alcohol in the human body. OKF4 and OKF6
were treated with acetaldehyde with effective
concentrations of 0 µM, 75 µM, 150 µM, 300
µM and 1000 µM in medium for a period of
48 hours with acetaldehyde added every 4
hours and media changed every 8 hours.
Quantitative RT-PCR
In vitro lncRNA expression was calculated by
method of qRT-PCR on the ethanol and
acetaldehyde treated cell lines with GAPDH
acting as the endogenous control for gene
expression.
Survival Data Analysis
Utilizing the original 34 patient cohort and
clinical information provided by the TCGA, we
correlated the expression levels of key
dysregulated lncRNAs with the long-term
survival of the patients. A Cox Proportional-
Hazards regression model was then utilized
to determine both univariate and multivariate
survival hazard ratios for the lncRNAs based
upon a low expression.
References
1) Heatmap depicting normalized lncRNA
expression levels (in the form of counts per
million) across the drinker and non-drinker
cohorts using 34 HNSCC patients. Top 100
differentially expressed lncRNAs shown,
including ones that do not have FDR < .05.
2) Panel of Differentially Expressed lncRNAs
with FDR between drinkers and nondrinkers.
NETO1-1 and lnc-SLC39A11-2 are
represented by different splice variants.
3) qRT-PCR analysis of ethanol-treated and
acetaldehyde cell lines, OKF4 and OKF6,
demonstrate that both PSD4-1 and NETO1-
1 are dysregulated by alcohol. Error bars
represent standard deviation.
4) Survival information for PSD4-1:14 in both
univariate and multivariate models
demonstrates a strong correlation with low
expression of PSD4-1:14 and better overall
survival.
5) Kaplan-Meier survival graph depicting a
correlation between low expression of lnc-
PSD4-1:14 and better survival.
Figure 1
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Figure 5