RNA-seq analysis identifies key long non-coding RNAs connected to the pathogenesis of alcohol-associated head and neck squamous cell carcinoma - PubMed (original) (raw)

RNA-seq analysis identifies key long non-coding RNAs connected to the pathogenesis of alcohol-associated head and neck squamous cell carcinoma

Vicky Yu et al. Oncol Lett. 2016 Oct.

Abstract

Alcohol consumption has been implicated in the pathogenesis of head and neck squamous cell carcinoma (HNSCC), although its mechanism is poorly understood. Recent advances in the identification and understanding of long non-coding RNAs (lncRNAs) have indicated that these molecules have a profound effect on numerous biological processes, including tumorigenesis and oncogenesis. The present authors hypothesize that alcohol-mediated dysregulation of lncRNAs is a key event in HNSCC pathogenesis. An in silico differential expression analysis utilizing RNA sequencing (RNA-seq) data from 34 HNSCC patients, which included alcohol drinkers and non-alcohol drinkers, identified a panel of lncRNAs that were dysregulated due to alcohol consumption. Normal oral keratinocytes were then exposed to ethanol and acetaldehyde to validate the RNA-seq results. Two lncRNAs that were differentially expressed due to alcohol consumption were identified from RNA-seq analysis of the clinical data: lnc-PSD4-1 and lnc-NETO-1. Oral keratinocytes exposed to alcohol and acetaldehyde demonstrated dysregulation of these two lncRNAs, thus validating the results of RNA-seq analysis. In addition, low expression of the lnc-PSD4-1 isoform, lnc-_PSD4_-1:14, exhibited a strong correlation with high survival rates in a Cox proportional hazards regression model. Therefore, these lncRNAs may 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.

Keywords: RNA-seq; acetaldehyde; alcohol; epigenetics; head and neck squamous cell carcinoma; long non-coding RNAs.

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Figures

Figure 1.

Figure 1.

(A) Cell proliferation and viability in alcohol-treated cells. Cell viability with increasing levels of ethanol concentration was measured in (B) OKF4 and (C) OKF6 cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Error bars denote standard deviation.

Figure 2.

Figure 2.

Heatmap of alcohol-dysregulated lncRNAs. Heatmap depicting normalized lncRNA expression levels (in the form of counts per million) across the alcohol drinker and non-alcohol drinker cohorts in 34 patients with head and neck squamous cell carcinoma. The top 100 differentially expressed lncRNAs are presented, including those whose false discovery rate was not <0.05. lnc, long non-coding.

Figure 3.

Figure 3.

Validation of alcohol-dysregulated lncRNAs identified in vitro with ethanol-treated cell lines. Reverse transcription-quantitative polymerase chain reaction analysis of ethanol-treated OKF4 and OKF6 cell lines demonstrated that (A) lnc-NETO1-1 and (B) lnc-PSD4-1 are dysregulated by alcohol. Error bars represent the standard deviation, as calculated by the 2−ΔΔCq method. lnc, long non-coding.

Figure 4.

Figure 4.

Validation of alcohol-dysregulated lncRNAs in vitro in acetaldehyde-treated cell lines. Reverse transcription-quantitative polymerase chain reaction analysis of acetaldehyde-treated OKF4 and OKF6 cell lines demonstrated that (A) lnc-NETO1-1 and (B) lnc-PSD4-1 are dysregulated by acetaldehyde. Error bars represent the standard deviation, as calculated by the 2−ΔΔCq method. lnc, long non-coding.

Figure 5.

Figure 5.

Kaplan-Meier survival curve for lnc-PSD4-1:14. Kaplan-Meier survival graph depicted a correlation between low expression levels of lnc-PSD4-1:14 and high survival in patients with head and neck squamous cell carcinoma. lnc, long non-coding.

References

    1. Hashibe M, Brennan P, Benhamou S, Castellsague X, Chen C, Curado MP, Dal Maso L, Daudt AW, Fabianova E, Fernandez L, et al. 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. 2007;99:777–789. doi: 10.1093/jnci/djk179. -DOI -PubMed
    1. Boffetta P, Hashibe M. Alcohol and cancer. Lancet Oncol. 2006;7:149–156. doi: 10.1016/S1470-2045(06)70577-0. -DOI -PubMed
    1. Homann N, Jousimies-Somer H, Jokelainen K, Heine R, Salaspuro M. High acetaldehyde levels in saliva after ethanol consumption: Methodological aspects and pathogenetic implications. Carcinogenesis. 1997;18:1739–1743. doi: 10.1093/carcin/18.9.1739. -DOI -PubMed
    1. Rahimy E, Kuo SZ, Ongkeko WM. Evaluation of non-coding RNAs as potential targets in head and neck squamous cell carcinoma cancer stem cells. Curr Drug Targets. 2014;15:1247–1260. doi: 10.2174/1389450115666141024113446. -DOI -PubMed
    1. Deng G, Sui G. Noncoding RNA in oncogenesis: A new era of identifying key players. Int J Mol Sci. 2013;14:18319–18349. doi: 10.3390/ijms140918319. -DOI -PMC -PubMed

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