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Characterization of selective exosomal microRNA expression profile derived from laryngeal squamous cell carcinoma detected by next generation sequencing

Huang, Qiang, Yang, Jiechao, Zheng, Juan, Hsueh, Chiyao, Guo, Yang, Zhou, Liang·2018·Oncology Reports
DOI 10.3892/or.2018.6672PMC PMC6151897
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Figure 1. Characterization of exosomes. (A) Electron microscopy of exosomes derived from the LSCC cell line AMC-HN-8. The image revealed membrane-bound particles that were homogeneous in appearance and ranged from 30–150 nm in size. Scale bar, 200 nm. (B) The diagram revealed the size distribution of exosomes by means of NTA. Error bars indicated +/-1 standard error of the mean. (C) Representative images of flow cytometry. Exosomes derived from the LSCC cell line AMC-HN-8 were stained with anti-CD63 and anti-CD81 immunofluorescence antibodies and analyzed by flow cytometry. Values indicate the ratio of positive exosomes. LSCC, laryngeal squamous cell carcinoma; NTA, nanoparticle tracking analysis.
Figure 1. Characterization of exosomes. (A) Electron microscopy of exosomes derived from the LSCC cell line AMC-HN-8. The image revealed membrane-bound particles that were homogeneous in appearance and ranged from 30–150 nm in size. Scale bar, 200 nm. (B) The diagram revealed the size distribution of exosomes by means of NTA. Error bars indicated +/-1 standard error of the mean. (C) Representative images of flow cytometry. Exosomes derived from the LSCC cell line AMC-HN-8 were stained with anti-CD63 and anti-CD81 immunofluorescence antibodies and analyzed by flow cytometry. Values indicate the ratio of positive exosomes. LSCC, laryngeal squamous cell carcinoma; NTA, nanoparticle tracking analysis.CC BY-NC-ND · PubMed Central
Figure 2. Comparison of total reads annotation in the cells and exosomes derived from LSCC cell line AMC-HN-8. (A) The total RNA read sequences in the cells and exosomes, revealed the annotation of ncRNA. The percentage is calculated as a percentage of clean reads. (B) The common reads of total RNA for cells and exosomes only accounted for 5.63%. (C) The diagram displayed the top 10 miRNAs in cells and exosomes, respectively. LSCC, laryngeal squamous cell carcinoma.
Figure 2. Comparison of total reads annotation in the cells and exosomes derived from LSCC cell line AMC-HN-8. (A) The total RNA read sequences in the cells and exosomes, revealed the annotation of ncRNA. The percentage is calculated as a percentage of clean reads. (B) The common reads of total RNA for cells and exosomes only accounted for 5.63%. (C) The diagram displayed the top 10 miRNAs in cells and exosomes, respectively. LSCC, laryngeal squamous cell carcinoma.CC BY-NC-ND · PubMed Central
Figure 3. The different microRNA expression profiles between parental cells and exosomes. (A) The heat map revealed the distinct miRNA expression profiles between parental cells and exosomes. The inclusion criteria was a 2-fold difference of log2 (fold-change) in either direction with a P-value <0.05. Red signal, high relative expression; green signal, low relative expression. (B) The volcano plot revealed the marked expression of miRNAs in exosomes. The inclusion criteria was a 2-fold difference of log2 (fold-change) in either direction with a P-value <0.05. Red, upregulated in the exosomes; green, downregulated in the exosomes.
Figure 3. The different microRNA expression profiles between parental cells and exosomes. (A) The heat map revealed the distinct miRNA expression profiles between parental cells and exosomes. The inclusion criteria was a 2-fold difference of log2 (fold-change) in either direction with a P-value <0.05. Red signal, high relative expression; green signal, low relative expression. (B) The volcano plot revealed the marked expression of miRNAs in exosomes. The inclusion criteria was a 2-fold difference of log2 (fold-change) in either direction with a P-value <0.05. Red, upregulated in the exosomes; green, downregulated in the exosomes.CC BY-NC-ND · PubMed Central
Figure 4. RT-qPCR validation. RT-qPCR was used to validate the results of small RNA sequencing. Six miRNAs accepted in the analysis (miR-320d, miR-1246, miR-122-5p, miR-4483, miR-30c-5p and let-7e-5p) were selected randomly. (A) LSCC cell line AMC-HN-8. (B) LSCC cell line Tu212. (C) LSCC cell line Tu686. The obtained values were normalized to hsa-miR-93-5p used as an internal control. Black, RNA-sequencing; Grey, RT-qPCR. RT-qPCR, reverse transcription quantitative PCR; LSCC, laryngeal squamous cell carcinoma.
Figure 4. RT-qPCR validation. RT-qPCR was used to validate the results of small RNA sequencing. Six miRNAs accepted in the analysis (miR-320d, miR-1246, miR-122-5p, miR-4483, miR-30c-5p and let-7e-5p) were selected randomly. (A) LSCC cell line AMC-HN-8. (B) LSCC cell line Tu212. (C) LSCC cell line Tu686. The obtained values were normalized to hsa-miR-93-5p used as an internal control. Black, RNA-sequencing; Grey, RT-qPCR. RT-qPCR, reverse transcription quantitative PCR; LSCC, laryngeal squamous cell carcinoma.CC BY-NC-ND · PubMed Central
Figure 5. Bioinformatics analysis of potential targets of selective exosomal miRNAs. (A) GO annotation of predicted targets. (B) KEGG pathway enrichment analysis of predicted targets. GO terms and KEGG pathway both with a P-value <0.05 were selected. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 5. Bioinformatics analysis of potential targets of selective exosomal miRNAs. (A) GO annotation of predicted targets. (B) KEGG pathway enrichment analysis of predicted targets. GO terms and KEGG pathway both with a P-value <0.05 were selected. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.CC BY-NC-ND · PubMed Central
Figure 6. Selective exosomal miRNA-gene co-expression network. (A) The network revealed the interactions between exosomal miRNAs and genes. Blue, miRNAs; Pink, genes. (B) The most common overlap of genes.
Figure 6. Selective exosomal miRNA-gene co-expression network. (A) The network revealed the interactions between exosomal miRNAs and genes. Blue, miRNAs; Pink, genes. (B) The most common overlap of genes.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Huang, Q., Yang, J., Zheng, J., Hsueh, C., Guo, Y., & Zhou, L.
Affiliations
Department of Otorhinolaryngology Head and Neck Surgery, Eye, Ear, Nose, and Throat Hospital, Fudan University, Shanghai 200031, P.R.�China
ORCID
Huang, Qiang
Venue
Oncology Reports
Publisher
Spandidos Publications
Published
2018-08-24
ISSN
  • 1021-335X
  • 1791-2431
DOI
10.3892/or.2018.6672
PMC
PMC6151897
Cited by
19
References
3

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