miRNA profiling in vitreous humor, vitreal exosomes and serum from uveal melanoma patients: Pathological and diagnostic implications

Abstract

Uveal melanoma (UM) represents approximately 5-6% of all melanoma diagnoses and up to 50% of patients succumb to their disease. Although several methods are available, accurate diagnosis is not always easily feasible because of potential accidents (e.g., intraocular hemorrhage). Based on the assumption that the profile of circulating miRNAs is often altered in human cancers, we verified whether UM patients showed different vitreous humor (VH) or serum miRNA profiles with respect to healthy controls. By using TaqMan Low Density Arrays, we analyzed 754 miRNAs from VH, vitreal exosomes, and serum of 6 UM patients and 6 healthy donors: our data demonstrated that the UM VH profile was unique and only partially overlapping with that from serum of the same patients. Whereas, 90% of miRNAs were shared between VH and vitreal exosomes, and their alterations in UM were statistically overlapped with those of VH and vitreal exosomes, suggesting that VH alterations could result from exosomal dysregulation. We report 32 miRNAs differentially expressed in UM patients in at least 2 different types of samples analyzed. We validated these data on an independent cohort of 12 UM patients. Most alterations were common to VH and vitreal exosomes (e.g., upregulation of miR-21,-34 a,-146a). Interestingly, miR-146a was upregulated in the serum of UM patients, as well as in serum exosomes. Upregulation of miR-21 and miR-146a was also detected in formalin-fixed, paraffin-embedded UM, suggesting that VH or serum alterations in UM could be the consequence of disregulation arising from tumoral cells. Our findings suggest the possibility to detect in VH and serum of UM patients "diagnostic" miRNAs released by the affected eye: based on this, miR-146a could be considered a potential circulating marker of UM.

Keywords: exosomes, microRNAs, serum, uveal melanoma, vitreous humor.

Figure 1.

Characterization of VH exosomes. (A) Average particle size in VH exosome samples was determined by dynamic light scattering. Y-axes: signal intensity (%); X-axes: size of particles (nm). (B) Flow cytometry detection of surface molecules on nanoparticles isolated from VH samples. The exosomes were bound to aldehyde-sulfate latex beads conjugated with anti-CD9, anti-CD63 or anti-CD81 antibodies and analyzed by flow cytometry. The antibodies were compared with their appropriate isotype control IgG1.

Figure 2.

Comparison of miRNAs found in VH, VH exosomes and serum of UM and healthy controls. (A) Venn diagrams showing the overlap between miRNA sets found in different types of samples. (B) Quantitative representation of miRNA different expression between UM patients and controls in VH, VH exosomes, serum. (C) Correlation between RQs from VH and its exosomes: x-axis represents the −log10 of RQ of vitreal miRNAs in UM patients with respect to normal controls; y-axis represents the −log10 of RQ of exosomal miRNAs in UM patients with respect to normal controls.

Figure 3.

Single TaqMan assays for miR-21, miR-34a, miR-146a, miR-618. Box plots representing the expression of: (A) miR-146a, (B) miR-21, (C) miR-34a, (D) miR-618, analyzed by single TaqMan assay on whole vitreous humor, exosomes from vitreous (ExoVitr.), whole serum, or exosomes from serum (ExoSer.) from an independent cohort of 12 patients. y-axis represents the –ΔCt of miRNAs in UM patients with respect to normal controls. Statistical significance was evaluated by the Wilcoxon rank sum test (p-value < 0.05).

Figure 4.

MiRNAs expression in FFPE UM specimens. Box plots representing the expression of: (A) miR-146a, (B) miR-21, (C) miR-34a, analyzed by single TaqMan assay on paraffin-embedded UM compared to healthy choroidal melanocytes. y-axis represents the −ΔCt of miRNAs in UM patients with respect to normal controls. Statistical significance was evaluated by the Wilcoxon rank sum test (p-value < 0.05).