Serum Exosomal miR-941 as a promising Oncogenic Biomarker for Laryngeal Squamous Cell Carcinoma

Abstract

At present, no blood-based biomarkers have been used in clinical practice for laryngeal squamous cell carcinoma (LSCC). Increasing evidence suggests that circulating exosomal microRNAs (miRNAs) may serve as potential diagnostic biomarkers for various cancers. This study aims to identify and evaluate serum exosomal miRNAs for LSCC diagnosis. The ExoQuick solution (EQ), which provides a high-yield and is a highly efficient exosome isolation method, was selected to isolate serum exosomes in the current study. In LSCC samples, exosome concentrations were higher than in healthy control (HC) samples. RNA-seq analysis identified a total of 1608 miRNAs, with 34 upregulated and 41 downregulated in LSCC samples relative to HC samples. Furthermore, qRT-PCR showed that miR-941 is significantly upregulated in LSCC serum exosomes, with this same trend seen in LSCC tissues and cells. Moreover, when examining miR-941 in cell lines, miR-941 overexpression promoted proliferation and invasion, while miR-941 knockdown inhibited cell proliferation and invasion. ROC curve analysis showed that miR-941 has an area under the curve (AUC) of 0.797 (95% CI = 0.676-0.918) for distinguishing LSCC patients from HCs. In conclusion, serum exosomal miR-941 may serve as a promising oncogenic biomarker for diagnosing LSCC, and has the potential as a therapeutic target.

Keywords: biomarker; diagnosis; exosome; hsa-miR-941; laryngeal squamous cell carcinoma.

Figure 1

Characterization and miRNA Profiles for Three LSCC Serum Exosomal Samples Isolated with Four Different Isolation Methods. (A) Representative transmission electron microscope (TEM) images for each extraction method. Scale bars = 50 nm. (B) Representative size distribution profiles obtained with Nanoparticle Tracking Analysis (NTA). (C) Modal sizes (nm) and (D) concentrations (particles/mL) of exosome samples examined via NTA. (E) Western blot analysis of exosomal markers (CD81, TSG101 and CD63) in lysates obtained by using the four different isolation methods. (F) Scatterplots of miRNAs RNA-seq expression profiles. Pearson correlation coefficient (r) was used as a measure of the strength of the linear relationship between the two exosomes samples obtained with two different methods. (G) Venn diagram showing unique and shared miRNAs between the UC, EQ, PEG1 and PEG2 samples.

Figure 2

LSCC Serum Exosome Levels are Higher Relative to the HC Samples. (A) Representative Exo-Check Exosome Antibody Array for detecting exosomal markers (CD81, CD63, ALIX, FLOT1, ICAM, EpCam, ANXA5 and TSG101) and assessing cellular contamination (cis-Golgi matrix-associated protein GM130). (B) Representative TEM images of serum exosomes in LSCC and HC samples. Red arrows indicate exosomes, scale bars = 0.2 µm. (C) Serum exosome concentration (particles/mL) in LSCC and HC samples as detected with NTA. Data are presented as a mean ± standard deviation (SD). *P < 0.05.

Figure 3

RNA-seq Analysis of the Discovery Set Including 6 LSCC and 6 HC Samples. (A) Analysis of serum exosomal RNA using an Agilent 2100 and electrophoresis indicated a significant population of small RNAs and an absence of 18S and 28S RNAs. (FU, fluorescence units; nt, nucleotides). (B) Length distribution of sequenced small RNAs (sRNAs). (C) Number of 18-35 nt sRNA reads vs. number of mapped reads. The horizontal and vertical lines are the mean levels of mapped reads and sRNA reads, respectively. (D) Hierarchical clustering of the differentially expressed LSCC and HC miRNAs. (E) Volcano plot identifying 34 significantly upregulated (red dots) and 41 downregulated (green dots) miRNAs. The horizontal line represents a P-value of 0.05, with the differentially expressed cut-off threshold set to P < 0.05 and | log2 fold-change | ≥ 0.5. (F) KEGG pathway analysis scatter plot of the predicted target genes associated with the differentially expressed miRNAs.

Figure 4

Quantitative Analyses of miRNAs in Clinical Samples and Receiver Operating Characteristic (ROC) Curve Analysis. (A) Candidate endogenous references selected from the discovery set (n = 12) RNA-seq data. The scatter plot shows expression level distributions normalized to TPM (transcript per million) and the coefficient of variation (CV) values. The y-axis represents the mean miRNA expression levels and is displayed as log10 (mean TPM). The x-axis represents the dispersion degree of these miRNAs and is described by the CV. The most stably expressed miRNAs with a moderate expression are indicated with red dots (n = 8). The blue dotted line represents the maximum CV value of these 8 miRNAs (0.236). (B) A subset of 9 differentially upregulated miRNAs identified from the RNA-seq data were further examined using an independent validation set (50 LSCC patients and 25 HCs), with expression levels visualized using box plots. MiRNA expression levels were detected using qRT-PCR, with selected endogenous references (miR-30a-5p, miR-532-5p and U6) used as controls. The Y-axis displays the expression level as log10(2^-ΔCt). *** P < 0.001, ** P < 0.01. (C) ROC curves assessing LSCC miR-941(blue line) and miR-27a-5p (green line), with area under the curve (AUC) values also determined. (D) Kaplan-Meier survival curves examining miR-941 expression in head and neck squamous cell carcinoma (HNSCC) tissue sample data obtained from the TCGA database. MiR-941 was significantly correlated with a poor outcome (log rank test, P = 0.0016). (E) Examination of HNSCC primary solid tumor data obtained from the YM500v3 database showed increased miR-941 expression. (F) Expression levels of miR-941 in different tumors from the YM500v3 database. The Y-axis represents the relative expression level in the tumors as compared to normal tissues. The X-axis displays the different tumor types. BLCA, Bladder urothelial carcinoma; BRCA, Breast invasive carcinoma; CESC, Cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD, Colon adenocarcinoma; ESCA, Esophageal carcinoma; HNSC, Head and Neck squamous cell carcinoma; KICH, Kidney chromophobe; KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma; PAAD, Pancreatic adenocarcinoma; PRAD, Prostate adenocarcinoma; READ, Rectum adenocarcinoma; SKCM, Skin cutaneous melanoma; STAD, Stomach adenocarcinoma.

Figure 5

miR-941 is Highly Expressed in LSCC Cell Lines and Promotes Proliferation and Invasion. (A) Cellular and exosomal RNA was extracted from two LSCC cell lines (FD-LSC-1 and Tu 686) and a normal cell line (HOK), and miR-941 levels were quantified via qRT-PCR. Data were normalized to levels of U6 and compared with the nontumor cell line HOK. (B) MiR-941 expression was detected in FD-LSC-1 and Tu 686 by qRT-PCR after transfection of miR-941 mimics, miR-941 inhibitor, or associated controls. (C) A CCK8 assay was performed to determine the effect of miR-941 gain or loss on cell proliferation. (D) The effect of miR-941 gain or loss on cell invasion was evaluated by using a Transwell invasion assay. Representative images are shown. Cell numbers were counted by randomly selecting five fields at 100× magnification. Data are presented as a mean ± SD. ***P < 0.001, **P < 0.01, *P < 0.05.