Bioinformatics analysis of aberrantly expressed exosomal lncRNAs in oral squamous cell carcinoma (CAL-27 vs. oral epithelial) cells

Abstract

Oral squamous cell carcinoma (OSCC) is the most prevalent form of malignant tumour in the oral cavity and its early detection is critical for improving the prognosis of affected patients. The present study aimed to isolate and confirm exosomes derived from the supernatant of the OSCC cell line CAL-27 and human oral epithelial cells (HOECs), analyze long non-coding RNA (lncRNA) expression profiles and determine the diagnostic value based on bioinformatics analyses. The results indicated that the particles isolated from the supernatant of CAL-27 and HOECs were either round or oval, had a size range of 30-150 nm and were enriched with ALG-2 interacting protein X (ALIX) and tumour susceptibility 101 proteins (TSG101). These characteristics confirmed that these particles were exosomes. Three lncRNAs (NR-026892.1, NR-126435.1 and NR-036586.1) were selected as potential diagnostic biomarkers for OSCC. The expression levels of the selected lncRNAs were significantly different in CAL-27-exo vs. HOEC-exo, as well as in whole cells (CAL-27 vs. HOECs) (P<0.001). The expression levels of the three lncRNAs confirmed by quantitative PCR were consistent with the sequencing data. In conclusion, various lncRNAs were aberrantly expressed between cancerous and non-cancerous exosomes, suggesting that they may serve as biomarkers for cancer.

Keywords: bioinformatics; exosome; long non-coding RNAs; oral squamous cell carcinoma.

Figure 1.

Exosome identification. (A) Western blot analysis of exosome markers (samples with different exposures); (B) transmission electron microscopy images of exosomes (magnification, ×30,000; scale bar, 200 nm); (C) Zetasizer Nano ZS analysis of the mean size of exosomes (~100 nm). ALIX, ALG-2 interacting protein X; TSG101, tumor susceptibility 101; HOEC, human oral epithelial cell; exo, exosome; d, diameter.

Figure 2.

Hierarchical clustering and volcano plots of differentially expressed ncRNAs and lncRNAs between HOEC-exo and CAL-27-exo. (A) Hierarchical clustering analysis of differentially expressed ncRNAs; each column represents a sample and each row indicates an lncRNA. The colour indicates the value of log₁₀ (RPKM+1). (B) Volcano plot of differentially expressed ncRNAs; the abscissa refers to the fold-change of ncRNA and lncRNA expression in different samples and the ordinate corresponds to the significance of the differential expression of the ncRNAs and lncRNAs. (C) Hierarchical clustering analysis of differentially expressed lncRNAs; each column represents a sample and each row indicates an lncRNA. The colour indicates the value of log₁₀ (RPKM+1). (D) Volcano plot of differentially expressed lncRNAs; the abscissa refers to the fold-change of ncRNA and lncRNA expression in different samples and the ordinate corresponds to the significance of the differential expression of the ncRNAs and lncRNAs. Red dots denote significant upregulation of ncRNAs and lncRNAs and green dots represent significant downregulation of ncRNAs and lncRNAs. HOEC, human oral epithelial cell; lncRNA, long non-coding RNA; exo, exosome; RPKM, Reads Per Kilobase per Million mapped reads.

Figure 3.

GO terms and KEGG pathway analysis with respect to the target genes of lncRNAs. (A-C) GO annotation in the categories (A) biological process, (B) cellular component and (C) molecular function. (D) KEGG pathways. GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long non-coding RNA.

Figure 4.

Network of three lncRNA-miRNA interactions. (A) NR_026892.1, (B) NR_126435.1 and (C) NR_036586.1. Red squares represent lncRNAs, blue circles correspond to mRNAs and pink lines refer to the target interaction of lncRNA-miRNA. lncRNA, long non-coding RNA; miRNA/miR, microRNA; hsa, Homo sapiens.

Figure 5.

Relative expression levels of NR-026892.1, NR-126435.1 and NR-036586.1 in (A) exosomes (n=3), (B) cells (n=3) and (C) sequencing data (n=1). HOEC, human oral epithelial cell; exo, exosome; lncRNA, long non-coding RNA.