miRNAs derived from circulating small extracellular vesicles as diagnostic biomarkers for nasopharyngeal carcinoma

Abstract

The microRNAs (miRNAs) in circulating small extracellular vesicles (sEVs) have been suggested as potential biomarkers in cancer diagnosis. This study was designed to evaluate the circulating sEV-derived miRNAs as biomarkers for the diagnosis of nasopharyngeal carcinoma (NPC). We compared the miRNA profiles in plasma-derived sEVs between 16 patients with NPC and 5 healthy controls (HCs). A distinct set of miRNAs that were differentially expressed between patients with NPC and HCs was determined by means of integrative bioinformatics approaches. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis revealed that the target genes of the differentially expressed miRNAs (DEMs) were mainly involved in cancer-associated signaling pathways. Seven representative DEMs were selected and further validated in an additional 60 patients with NPC and 40 HCs using quantitative reverse-transcription PCR analysis (qRT-PCR). Receiver operating characteristic (ROC) curve analysis was used to assess the accuracy of the sEV-miRNA-based model for diagnosis. The 3 miRNA-based model, comprising miR-134-5p, miR-205-5p, and miR-409-3p, showed good discriminating power with an area under the curve (AUC) value of 0.88 in the training set and 0.91 in the validation set. Furthermore, the diagnostic model had an excellent classification ability to distinguish patients with NPC at different clinical stages or Epstein-Barr virus infection status from HCs. In conclusion, our findings indicated that sEV-derived miRNA levels were altered in the plasma of patients with NPC in comparison with those in HCs. The model based on the 3 sEV-derived miRNAs could potentially act as an alternative or complementary approach for diagnosing NPC.

Keywords: biomarker; diagnosis; microRNA; nasopharyngeal carcinoma; small extracellular vesicle.

FIGURE 1

Characteristics of plasma‐derived sEVs. A, Images of transmission electron microscopy showing that sEVs are bowl shaped. Scale, 200 nm. B, Western blot analysis showing the expression of CD63, TSG101, and Alix, and the absence of calnexin. C, Nanoparticle tracking analysis showing the size distribution of sEVs; mean size was 144.6 nm. CL, cell lysates

FIGURE 2

Characterization of plasma‐derived sEV‐miRNAs in patients with NPC. A, Sequencing saturation analysis. The number of effective miRNAs (TPM > 10) detected in the sequencing data in each sample. B, Distribution of mappable small RNAs by NGS. C, Heatmap of differentially expressed miRNAs in plasma sEVs. D, Bubble diagram for analysis of genes targeted by the differentially expressed miRNAs in KEGG analysis. scRNA, small cytoplasmic RNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; Repbase, repeat sequence; tRNA, transfer RNA; rRNA, ribosomal RNA; miRNA, microRNA; NPC, nasopharyngeal carcinoma; HC, healthy control

FIGURE 3

UpSet intersection diagram. Five comparisons are shown in the UpSet plot. The numbers on the bars show the numbers of overlapping miRNAs in the corresponding comparison

FIGURE 4

Signaling network diagram for the prediction of genes targeted by the 7 candidate miRNAs

FIGURE 5

Expression levels of selected miRNAs using qRT‐PCR in the validation set. A, miR‐134‐5p; B, miR‐486‐5p; C, miR‐486‐3p; D, miR‐205‐5p; E, miR‐409‐3p; F, miR‐484; G, miR‐92b‐3p. H, I, Subgroup analysis of the expression of miR‐205‐5p in patients with NPC and HCs. miR‐205‐5p expression was significantly higher in patients with advanced stage NPC (H) or EBV‐positive infection (I) than that in HCs. NPC, nasopharyngeal carcinoma; HC, healthy control

FIGURE 6

Construction and validation of the diagnostic model based on the 3 miRNAs in the qPCR validation cohort. A, ROC curves of the 3 miRNA (miR‐134‐5p, miR‐205‐5p, miR‐409‐3p)‐based model in the training and validation sets. B, ROC curves for the diagnosis of patients with NPC using the 3 miRNA‐based model in the subgroup analysis. Risk probability distribution plots and heatmap of the 3 miRNAs in the training set (C), and validation set (D), NPC, nasopharyngeal carcinoma; HC, healthy control; EB, patients with NPC positive for Epstein‐Barr virus infection; NEB, patients with NPC negative for Epstein‐Barr virus infection; Early, patients with NPC diagnosed at an early stage; Advanced, patients with NPC diagnosed at an advanced stage