Transcriptome sequencing uncovers novel long noncoding and small nucleolar RNAs dysregulated in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma persists as one of the most common and deadly malignancies, with early detection and effective treatment still posing formidable challenges. To expand our currently sparse knowledge of the noncoding alterations involved in the disease and identify potential biomarkers and therapeutic targets, we globally profiled the dysregulation of small nucleolar and long noncoding RNAs in head and neck tumors. Using next-generation RNA-sequencing data from 40 pairs of tumor and matched normal tissues, we found 2808 long noncoding RNA (lncRNA) transcripts significantly differentially expressed by a fold change magnitude ≥2. Meanwhile, RNA-sequencing analysis of 31 tumor-normal pairs yielded 33 significantly dysregulated small nucleolar RNAs (snoRNA). In particular, we identified two dramatically down-regulated lncRNAs and one down-regulated snoRNA whose expression levels correlated significantly with overall patient survival, suggesting their functional significance and clinical relevance in head and neck cancer pathogenesis. We confirmed the dysregulation of these noncoding RNAs in head and neck cancer cell lines derived from different anatomic sites, and determined that ectopic expression of the two lncRNAs inhibited key EMT and stem cell genes and reduced cellular proliferation and migration. As a whole, noncoding RNAs are pervasively dysregulated in head and squamous cell carcinoma. The precise molecular roles of the three transcripts identified warrants further characterization, but our data suggest that they are likely to play substantial roles in head and neck cancer pathogenesis and are significantly associated with patient survival.

Identification and characterization of dysregulated P-element induced wimpy testis-interacting RNAs in head and neck squamous cell carcinoma.

It is clear that alcohol consumption is a major risk factor in the pathogenesis of head and neck squamous cell carcinoma (HNSCC); however, the molecular mechanism underlying the pathogenesis of alcohol-associated HNSCC remains poorly understood. The aim of the present study was to identify and characterize P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) and PIWI proteins dysregulated in alcohol-associated HNSCC to elucidate their function in the development of this cancer. Using next generation RNA-sequencing (RNA-seq) data obtained from 40 HNSCC patients, the piRNA and PIWI protein expression of HNSCC samples was compared between alcohol drinkers and non-drinkers. A separate piRNA expression RNA-seq analysis of 18 non-smoker HNSCC patients was also conducted. To verify piRNA expression, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed on the most differentially expressed alcohol-associated piRNAs in ethanol and acetaldehyde-treated normal oral keratinocytes. The correlation between piRNA expression and patient survival was analyzed using Kaplan-Meier estimators and multivariate Cox proportional hazard models. A comparison between alcohol drinking and non-drinking HNSCC patients demonstrated that a panel of 3,223 piRNA transcripts were consistently detected and differentially expressed. RNA-seq analysis and in vitro RT-qPCR verification revealed that 4 of these piRNAs, piR-35373, piR-266308, piR-58510 and piR-38034, were significantly dysregulated between drinking and non-drinking cohorts. Of these four piRNAs, low expression of piR-58510 and piR-35373 significantly correlated with improved patient survival. Furthermore, human PIWI-like protein 4 was consistently upregulated in ethanol and acetaldehyde-treated normal oral keratinocytes. These results demonstrate that alcohol consumption may cause dysregulation of piRNA expression in HNSCC and in vitro verifications identified 4 piRNAs that may be involved in the pathogenesis of alcohol-associated HNSCC.

The non-coding landscape of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease marked by frequent recurrence and metastasis and stagnant survival rates. To enhance molecular knowledge of HNSCC and define a non-coding RNA (ncRNA) landscape of the disease, we profiled the transcriptome-wide dysregulation of long non-coding RNA (lncRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA) using RNA-sequencing data from 422 HNSCC patients in The Cancer Genome Atlas (TCGA). 307 non-coding transcripts differentially expressed in HNSCC were significantly correlated with patient survival, and associated with mutations in TP53, CDKN2A, CASP8, PRDM9, and FBXW7 and copy number variations in chromosomes 3, 5, 7, and 18. We also observed widespread ncRNA correlation to concurrent TP53 and chromosome 3p loss, a compelling predictor of poor prognosis in HNSCCs. Three selected ncRNAs were additionally associated with tumor stage, HPV status, and other clinical characteristics, and modulation of their expression in vitro reveals differential regulation of genes involved in epithelial-mesenchymal transition and apoptotic response. This comprehensive characterization of the HNSCC non-coding transcriptome introduces new layers of understanding for the disease, and nominates a novel panel of transcripts with potential utility as prognostic markers or therapeutic targets.