Plasma-derived exosomal miR-4732-5p is a promising noninvasive diagnostic biomarker for epithelial ovarian cancer

Abstract

Background: Exosomal miRNAs regulate gene expression and play important roles in several diseases. We used exosomal miRNA profiling to investigate diagnostic biomarkers of epithelial ovarian cancer (EOC).

Methods: In total, 55 individuals were enrolled, comprising healthy (n = 21) and EOC subjects (n = 34). Small mRNA (smRNA) sequencing and real-time PCR (RT-PCR) were performed to identify potential biomarkers. Receiver operating characteristic (ROC) curves were conducted to determine biomarker sensitivity and specificity.

Results: Using smRNA sequencing, we identified seven up-regulated (miR-4732-5p, miR-877-5p, miR-574-3p, let-7a-5p, let-7b-5p, let-7c-5p, and let-7f-5p) and two down-regulated miRNAs (miR-1273f and miR-342-3p) in EOC patients when compared with healthy subjects. Of these, miR-4732-5p and miR-1273f were the most up-regulated and down-regulated respectively, therefore they were selected for RT-PCR analysis. Plasma derived exosomal miR-4732-5p had an area under the ROC curve of 0.889, with 85.7% sensitivity and 82.4% specificity in distinguishing EOC patients from healthy subjects (p<0.0001) and could be a potential biomarker for monitoring the EOC progression from early stage to late stage (p = 0.018).

Conclusions: Plasma derived exosomal miR-4732-5p may be a promising candidate biomarker for diagnosing EOC.

Keywords: Epithelial ovarian cancer; Exosomal miRNA profiling; Exosomes; miR-4732-5p; microRNAs.

Fig. 1

Identification of exosomes from plasma. a The black arrows indicate typical exosomes with size and morphology confirmed by TEM. Images were taken at 105,000 × magnification (scale bar = 100 nm). b Exosome particles from plasma were analyzed by NTA

Fig. 2

SmRNA sequence profiling and composition after data quality checks. Profiles of exosomal miRNAs after data quality checks. a smRNA composition of each sample, which means the ratio of smRNA class type. b Low expressed miRNAs were filtered by miRNA profiling. All transcript counts were filtered with more than zero in at least 50% (n ≥ 5) of all samples while miRNAs with zero counts in at least 50% samples are excluded

Fig. 3

Differential expression and miRNA profiling of plasma derived exosomes from six ovarian cancer patients compared to four healthy controls. a Volcano plot depicting differential expression results for miRNAs in ovarian tumor samples when compared with healthy controls. Threshold:|Log₂fold change | ≥ 1, p ≤ 0.05. b Heat-maps for differentially expressed miRNAs with a cut-off less than |Log₂fold change | ≥ 1, p ≤ 0.05, FDR ≤ 0.05

Fig. 4

Plasma derived exosomal miR-4732-5p as a diagnostic biomarker. SYBER green quantitative RT-PCR indicated that miR-4732-5p (a) was significantly up-regulated in ovarian cancer patient plasma derived exosomes when compared with healthy controls (p = 0.003),while there was no significant changed for miR-1273f (b). c Plasma derived exosomal miR-4732-5p was significantly up-regulated in late-stage ovarian cancer patient when compared with early-stage group (p = 0.016). d ROC curve analysis indicated that the AUC of miR-4732-5p between ovarian cancer patients and healthy controls was 0.889 (p<0.0001). e The correlation between CA-125 level and miR-4732-5p