Unraveling the oral cancer lncRNAome: Identification of novel lncRNAs associated with malignant progression and HPV infection

Abstract

Objectives: The role of long non-coding RNA (lncRNA) expression in human head and neck squamous cell carcinoma (HNSCC) is still poorly understood. In this study, we aimed at establishing the onco-lncRNAome profiling of HNSCC and to identify lncRNAs correlating with prognosis and patient survival.

Materials and methods: The Atlas of Noncoding RNAs in Cancer (TANRIC) database was employed to retrieve the lncRNA expression information generated from The Cancer Genome Atlas (TCGA) HNSCC RNA-sequencing data. RNA-sequencing data from HNSCC cell lines were also considered for this study. Bioinformatics approaches, such as differential gene expression analysis, survival analysis, principal component analysis, and Co-LncRNA enrichment analysis were performed.

Results: Using TCGA HNSCC RNA-sequencing data from 426 HNSCC and 42 adjacent normal tissues, we found 728 lncRNA transcripts significantly and differentially expressed in HNSCC. Among the 728 lncRNAs, 55 lncRNAs were significantly associated with poor prognosis, such as overall survival and/or disease-free survival. Next, we found 140 lncRNA transcripts significantly and differentially expressed between Human Papilloma Virus (HPV) positive tumors and HPV negative tumors. Thirty lncRNA transcripts were differentially expressed between TP53 mutated and TP53 wild type tumors. Co-LncRNA analysis suggested that protein-coding genes that are co-expressed with these deregulated lncRNAs might be involved in cancer associated molecular events. With consideration of differential expression of lncRNAs in a HNSCC cell lines panel (n=22), we found several lncRNAs that may represent potential targets for diagnosis, therapy and prevention of HNSCC.

Conclusion: LncRNAs profiling could provide novel insights into the potential mechanisms of HNSCC oncogenesis.

Keywords: Head and neck squamous cell carcinoma (HNSCC); Human papilloma virus (HPV); Long non-coding RNA; RNA-sequencing; TCGA; TP53.

Figure 1

Differential expression analysis between tumor and normal tissues in the HNSCC TCGA. (A) Heatmap of 728 differentially expressed lncRNAs in comparison between tumor and normal tissues. (B) Representative differentially expressed lncRNAs between tumor and normal tissues. (C) Gene Ontology (GO) enrichment analysis for protein-coding genes co-expressed with 728 lncRNAs with visualization by REViGO algorism.

Figure 2

Overall survival (OS) and disease-free survival (DFS) plots and functional annotation of co-expressed protein-coding genes which are neighbor of 55 prognosis-associated lncRNAs. (A) Representative Kaplan-Meier plots were shown in OS (upper row), and DFS (lower row). (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis is shown in bar chart for protein-coding genes co-expressed with 55 prognosis-associated lncRNAs.

Figure 3

Differential expression analysis between HPV status in the OPC-22 panel and the TCGA HNSCC. (A) Principal component analysis (PCA) among NOKSI as control cells (green), HPV+ cells (blue) and HPV- cells (pink). (B) Volcano plots shows the means difference of expression in HNSCC cells vs. control cells in X axis, and significance in HNSCC cells vs. control cells in Y axis. Broken line indicates p-value < 0.05. (C) Heatmap of 27 differentially expressed lncRNAs in comparison between HPV+ and HPV- cells. LncRNAs in bold are commonly up-modulated in HPV+ cells and tumors in TCGA HNSCC. (D) Heatmap of 140 differentially expressed lncRNAs in comparison between HPV+ and HPV- tumors in TCGA.

Figure 4

Differential expression analysis based on TP53 mutation status in the HNSCC TCGA. (A) Heatmap of 42 differentially expressed lncRNAs in comparison between TP53 mutation and TP53 wild type. Sub organ site information (Pharynx: blue, Larynx: red, and Oral: green) and HPV infection status information (HPV+: dark gray, and HPV-: light blue) are included. (B) Venn diagram shows 19 overlapping up-modulated lncRNAs both in HPV+ and TP53 wild type tumors. (C) Representative up-modulated lncRNAs in TP53 wild type tumors are shown in bar chart. LncRNAs whose bars are in blue are significantly enriched in KEGG-P53 signaling pathway. (D) Co-expressed genes with LINC01315 or LINC00925 in KEGG-P53 signaling pathway (LINC01315: pink, and LINC00925: blue).