Comprehensive analysis of miRNA and protein profiles within exosomes derived from canine lymphoid tumour cell lines

Abstract

Exosomes are small extracellular vesicles released from almost all cell types, which play roles in cell-cell communication. Recent studies have suggested that microenvironmental crosstalk mediated by exosomes is an important factor in the escape of tumour cells from the anti-tumour immune system in human haematopoietic malignancies. Here, we conducted comprehensive analysis of the miRNA and protein profiles within the exosomes released from four canine lymphoid tumour cell lines as a model of human lymphoid tumours. The results showed that the major miRNAs and proteins extracted from the exosomes were similar among the four cell lines. However, the miRNA profiles differed among the exosomes of each cell line, which corresponded to the expression patterns of the parent cells. In the comparison of the amounts of miRNAs and proteins among the cell lines, those of three miRNAs (miR-151, miR-8908a-3p, and miR-486) and CD82 protein differed between exosomes derived from vincristine-sensitive and resistant cell lines. Further investigations are needed to elucidate the biological functions of the exosomal contents in the microenvironmental crosstalk of lymphoid tumours.

Fig 1

Hierarchical clustering (a) and PCA plots (b) for miRNA profiles of exosomes and parent cells of CLBL-1, GL-1, and UL-1. Exosomes and parent cells clustered similarly for each cell line and the profiles were different among cell lines. Orange dots (exosomes) and red dots (parent cells) correspond to CLBL-1, violet dots (exosomes) and blue dots (parent cells) to GL-1, and grey dots (exosomes) and black dots (parent cells) to UL-1.

Fig 2. Heat maps showing miRNAs whose amounts were significantly different between exosomes and parent cells.

The amounts of 39, 20, and 24 miRNAs were significantly different in CLBL-1 (a), UL-1 (b), and Ema (c), respectively (q < 0.01).

Fig 3. Comparison of the amounts of miR-350, miR-671, miR-22, and miR-8865 between exosomes and parent cells.

The amount of miR-350 (a) is significantly higher in exosomes compared with parent cells, whereas those of miR-22 (c), miR-671 (b), and miR-8865 (d) were not significantly different. All data represent the mean ± SD of three independent experiments. *P < 0.05.

Fig 4. Comparison of the amounts of miR-151, miR-8908a-3p, and miR-486 between VCR-S and VCR-R cell lines.

The amounts of miR-151 (a) and miR-8908a-3p (b) in VCR-S cell lines were significantly lower than in VCR-R cell lines, and that of miR-486 (c) in VCR-S cell lines were significantly higher than in VCR-R cell lines. All data represent the mean ± SD of three independent experiments. *P < 0.05.

Fig 5

Western blotting for CD82 using proteins extracted from exosomes (a) and parent cells (b) of each cell line. HSP90B and β-actin were selected for internal control for exosomes and parent cells, respectively. CD82 protein is detected in the exosomes of CLBL-1 and GL-1, whereas it was not detected in parent cells in any of the four cell lines. The figures that shows the detection of CD82 within exosomes and parent cells were cropped from the different parts of the same figure of the membrane. The figures of HSP90B and β-actin were cropped from the figures of the different membrane. The full-length figures of blotting membrane are shown in S7 Fig.