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

Figures & data

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).

Table 2. Demographics, tumor parameters, time treatment/enucleation in UM

Sex	Age	Location	Thickness (mm)	Largest diameter (mm)	Cell type	Extrascleral extension	Metastasis	Pathological T stage	miRNA Analysis
F	49	choroid/cil.body	15.93	19.7	mixed	N	N	pT4b	TLDA
M	67	choroid	16.27	20.8	spindle	N	N	pT4b	TLDA
F	54	choroid/cil.body	11.8	20.1	mixed	N	N	pT4b	TLDA
F	67	choroid/cil.body	15	20	spindle	N	N	pT4b	TLDA
F	67	choroid	11	17.5	mixed	N	N	pT3b	TLDA
F	29	choroid	8	11	mixed	N	N	pT2a	TLDA
F	67	choroid	14.5	16	spindle	N	N	pT4a	STA
F	72	choroid/cil.body	15.5	19.2	mixed	N	N	pT4b	STA
F	59	choroid	16.9	17	mixed	N	N	pT4a	STA
M	52	choroid	7.9	13.3	spindle	N	N	pT3a	STA
F	83	choroid/cil.body	5.8	15	mixed	N	N	pT2a	STA
M	48	choroid	5.5	10	spindle	N	N	pT2a	STA
M	54	choroid	9	13	spindle	N	N	pT3a	STA
F	74	choroid	4.88	13	mixed	N	N	pT2a	STA
F	37	choroid	4.6	10.2	mixed	N	N	pT2a	STA
M	64	choroid	2	7	Epithelioid	N	N	pT1a	STA
F	36	choroid	4.2	9	spindle	N	N	pT1a	STA
M	82	choroid	7.5	11.5	Epithelioid	N	N	pT2a	STA

TLDA: patients analyzed by TaqMan Low Density Arrays for VH, VH exosomes, serum.

STA: patients analyzed by Single TaqMan Assays for VH, VH exosomes, serum, serum exosomes, FFPE tissues.

Table 1. DE miRNAs in UM patients

DE miRNAs	RQ VH	RQ VH Exosomes	RQ Serum
let-7c	0.2	0.1	NDE
miR-16	3.7	4.4	NDE
miR-19a	3.3	2.6	0.2
miR-21	3.9	9.1	NDE
miR-30a-3p	0.7	0.4	NDE
miR-30d	21.0	NDE	0.1
miR-30e-3p	0.4	0.5	NDE
miR-34a	4.6	9.1	NDE
miR-93*	9.1	7.9	NDE
miR-126	3.1	7.0	NDE
miR-127	0.1	NDE	0.4
miR-130a	21.5	19.6	NDE
miR-139–5p	0.1	0.2	NDE
miR-146a	21.2	20.5	5.3
miR-149	0.1	0.1	NDE
miR-150	6.0	5.3	NDE
miR-155	0.1	0.04	NDE
miR-181a	0.1	0.2	NDE
miR-186	5.4	4.3	NDE
miR-378	8.2	5.5	NDE
miR-451	5.5	NDE	0.3
miR-483–5p	0.1	0.05	NDE
miR-486	2.6	5.6	NDE
miR-518f	NDE	0.3	0.4
miR-523	0.3	NDE	7.8
miR-618	0.2	3.5	NDE
miR-625*	0.2	0.3	NDE
miR-628–5p	0.2	0.5	NDE
miR-1227	0.5	0.3	NDE
miR-1255B	0.1	0.3	NDE
miR-1274A	0.6	8.9	NDE
miR-1274B	0.5	NDE	0.1

Differentially Expressed (DE) miRNAs in UM patients with respect to normal controls in at least 2 different kinds of biological samples analyzed. RQ: Relative Quantity. NDE: Not Differentially Expressed. False discovery rate <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).