Diagnostic and prognostic relevance of circulating exosomal miR-373, miR-200a, miR-200b and miR-200c in patients with epithelial ovarian cancer

Abstract

Exosomes are membrane vesicles that mediate intercellular communication by transporting their molecular cargo from cell to cell. We investigated whether serum levels of exosomal miR-373, miR-200a, miR-200b and miR-200c and circulating exosomes have diagnostic and prognostic relevance in a cohort of 163 epithelial ovarian cancer (EOC) patients using TaqMan MicroRNA assays and ELISA. The serum concentrations of exosomal miR-373 (p = 0.0001), miR-200a (p = 0.0001), miR-200b (p = 0.0001) and miR-200c (p = 0.028) were significantly higher in EOC patients than healthy women. The levels of miR-200a (p = 0.0001), miR-200b (p = 0.0001) and miR-200c (p = 0.019) could distinguish between malignant and benign ovarian tumors. While the levels of miR-373 and miR-200a were increased in all FIGO/lymph node stages (p = 0.0001), the levels of miR-200b and miR-200c were higher in patients with FIGO stage III-IV (p = 0.0001, p = 0.008, respectively) including lymph node metastasis (p = 0.0001, p = 0.004, respectively) than FIGO stages I-II. The increased levels of miR-200b and miR-200c were also associated with CA125 values (p = 0.0001, p = 0.0001, respectively) and a shorter overall survival (p = 0.007, p = 0.017, respectively). The levels of exosomes were excessively elevated in EOC patients (p = 0.0001). In all three cohorts, they were positively associated with the serum levels of exosomal miR-373 (p = 0.004), miR-200a (p = 0.0001), miR-200b (p = 0.0001) and miR-200c (p = 0.008). In conclusion, the increased levels of exosomal miR-200b and miR-200c mainly observed in advanced EOC suggest that these microRNAs may be involved in tumor progression. The high concentrations of exosomes in EOC patients imply an excessive, active exosomal secretion in EOC.

Keywords: diagnosis; epithelial ovarian cancer; exosomal miRNAs; exosomes; prognosis.

Figure 1. Workflow of the present study

Figure 2. Quantification of exosomal miR-373, miR-200a, miR-200b and miR-200c in the serum of healthy women, patients with benign ovarian diseases and EOC patients

Exosomes were extracted and pelleted from serum and analyzed by Western blot using antibodies specific for the exosome proteins Mucin1, CD63 and CD9. The Western blot shows a representative example of the exosome extraction of three EOC patients (A, above). Western Blot with non-lysed exosomes and an antibody specific for AGO2 protein. AGO2 protein is only detectable in HeLa cell protein that serves as a positive control (A, below). The box plot compares the exosomal miRNA concentrations in the serum of healthy women (n = 32), patients with benign ovarian diseases (n = 20) and EOC patients (n = 163) (B). ROC analyses show the profiles of sensitivity and specificity of exosomal miR-200a, miR-200b, miR-200c and their combination to distinguish benign ovarian diseases from EOC (C). Summarization of sensitivities and specificities of exosomal miR-373, miR-200a, miR-200b, miR-200c and their combination (D).

Figure 3. Correlations of the serum levels of exosomal miR-373, miR-200a, miR-200b and miR-200c with the clinical parameters of EOC patients

The box plot compares the exosomal miRNA concentrations in the serum of healthy women (n = 32) EOC patients with FIGO I-II (n = 27) and FIGO III-IV (n = 118) (A). The box plot compares the exosomal miRNA concentrations in the serum of healthy women (n = 32), lymph-node negative (N0, n = 48) and lymph-node positive (N1, n = 78) EOC patients (B). The scatter plots show the correlations of concentrations of exosomal miR-200b and miR-200c with the CA125 values of EOC patients (n = 77) (C).

Figure 4. Correlations of the serum levels of exosomal miR-373, miR-200b and miR-200c with overall survival and disease-free survival of EOC patients

Univariate Kaplan-Meier curves are related to low and high serum concentrations of exosomal miR-373 (A), miR-200b (B) and miR-200c (C) for overall survival and of miR-200c (D) for disease-free survival. The median values of each exosomal miRNA concentrations were used for grouping the EOC samples according to low (n = 46) and high (n = 47) transcript levels.

Figure 5. Increase in exosome levels in the serum of EOC patients and correlations between exosomes and exosomal miRNAs

The box plot compares the exosome levels in the serum of healthy women (n = 32), patients with benign ovarian diseases (n = 20) and EOC patients (n = 36) (A). The scatter plots show the correlations of exosomes with the levels of exosomal miR-373 (B), miR-200a (C), miR-200b (D) and miR-200c (E) in EOC patients, patients with benign ovarian diseases and heathy women (n = 88).