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. 2015;12(8):810-23.
doi: 10.1080/15476286.2015.1056975.

Small RNA Deep Sequencing Discriminates Subsets of Extracellular Vesicles Released by Melanoma cells--Evidence of Unique microRNA Cargos

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Free PMC article

Small RNA Deep Sequencing Discriminates Subsets of Extracellular Vesicles Released by Melanoma cells--Evidence of Unique microRNA Cargos

Taral R Lunavat et al. RNA Biol. .
Free PMC article

Abstract

Melanoma cells release different types of extracellular vesicles (EVs) into the extracellular milieu that are involved with communication and signaling in the tumor microenvironment. Subsets of EVs include exosomes, microvesicles, and apoptotic bodies that carry protein and genetic (RNA) cargos. To define the contribution of the RNA cargo of melanoma cell derived EVs we performed small RNA sequencing to identify different small RNAs in the EV subsets. Using validated centrifugation protocols, we separated these EV subsets released by the melanoma cell line MML-1, and performed RNA sequencing with the Ion Torrent platform. Various, but different, non-coding RNAs were detected in the EV subsets, including microRNA, mitochondrial associated tRNA, small nucleolar RNA, small nuclear RNA, Ro associated Y-RNA, vault RNA and Y-RNA. We identified in total 1041 miRNAs in cells and EV subsets. Hierarchical clustering showed enrichment of specific miRNAs in exosomes, including hsa-miR-214-3p, hsa-miR-199a-3p and hsa-miR-155-5p, all being associated with melanoma progression. Comparison of exosomal miRNAs with miRNAs in clinical melanoma samples indicate that multiple miRNAs in exosomes also are expressed specifically in melanoma tissues, but not in benign naevi. This study shows for the first time the presence of distinct small RNAs in subsets of EVs released by melanoma cells, with significant similarities to clinical melanoma tissue, and provides unique insights into the contribution of EV associated extracellular RNA in cancer.

Keywords: cancer; extracellular RNA; malignant melanoma; membrane vesicles; next-generation sequencing; non-coding RNA.

Figures

Figure 1.
Figure 1.
Characterization of extracellular vesicles including RNA profiles. Protein and RNA was extracted from MML-1 cells as well as apoptotic bodies, microvesicles and exosomes released by these cells. (A) Immunoblotting of the endoplasmic reticulum protein Calnexin, the mitochondrial protein Bcl-2, the nucleus protein nucleoporin p62 and the exosomal protein TSG101 and Flotillin-1 in isolates of EVs from MML-1 cells and cellular extract. All the antibodies were used in 1:1000 dilutions. (B) Characterization of EVs by Giemsa staining and transmission electron microscopy (TEM). Cytospin preparations were used for apoptotic bodies (50μg) and was stained with Giemsa. Scale bar 10μm. Ten microgram was used for the TEM for microvesicles and exosomes. Scale bar 200nm. (C) Bioanalyzer analysis of RNA isolated from cells, apoptotic bodies, microvesicles and exosomes on total RNA NanoChip and Small RNA chip. The rRNA peaks are indicated as 18S and 28S subunits in cells, apoptotic bodies and microvesicles, whereas exosomes shows reduced presence of rRNA. Cells and EVs all exhibited the miRNA region from 4-40nt whereas tRNA was enriched in cells, apoptotic bodies and microvesicles but absent in exosomes. These images are representative of 2 replicates, and the second replicate display a similar RNA profile. (D) The RNA yield is represented as ng (RNA)/million cells (n = 3). Data are presented as mean ± SEM. (E) Protein to RNA ratio in EVs is calculated by measuring the total amount of protein in micrograms (μg) and the total amount of RNA in micrograms (μg) per million cells. Data are presented as mean ± SEM. **P < 0.01, ns; non-significant.
Figure 2.
Figure 2.
For figure legend, See page 815. Figure 2. (see previous page) Enrichment of non-coding RNA in EVs. Relative enrichment of small non-coding RNA (A) small nucleolar RNA (snoRNA) (B) small nuclear RNA (snRNA) (C) mitochondrial associated RNA (D) Ro associated Y-RNA and (E) Miscellaneous RNA, compared to cells with negative binomial distribution. The data are presented as significant enrichment of non-coding RNA that is analyzed by DESeq2. A negative value means the enrichment of all noncoding RNA in EVs in relation to cells and positive value means the enrichment of all noncoding RNA in cells. (A) Enrichment of 47 small nucleolar RNA across all EVs. Snord89 and snord83A was enriched in exosomes and apoptotic bodies shown in negative fold expression. All other snoRNA was expressed in cells. (B) Small nuclear RNA enrichment in cells and EVs. There is presence of pseudogene snRNA enriched in all EVs and absent in cells. (C) Enrichment of 30 mitochondrial associated tRNA in cells and sub populations of EVs. Apoptotic bodies and microvesicles are enriched in most of the mtRNA and exosomes does not have any expression. (D and E) Enrichment of Ro associated y-RNA and miscellaneous RNA in EVs and absent in cells. All the data are the average of the duplicates analyzed from DESeq2. The data represented is the significant enrichment having P < 0.05.
Figure 3.
Figure 3.
Relationship of miRNA between cells and subsets of extracellular vesicles. (A) Principal component analysis was performed on the 8 samples using MATLAB (4 sample types with 2 biological replicates), presenting each sample replicates lying in each individual component. (B) Scatter plots showing correlation between the average expressions of the 2 biological replicates. All the correlation curves were visualized and calculated using MATLAB. 1 - EXO vs ABs Rs = 0.7934; 2 – MVs vs ABs Rs = 0.9064; 3 – Cells vs ABs Rs = 0.8885; 4 – MVs vs EXO Rs = 0.8628; 5 – Cells vs EXO Rs = 0.7482; 6 – Cells vs MVs Rs = 0.8642. False Discovery Rate (FDR) < 1%. ABs – Apoptotic Bodies, MVs – Microvesicles, EXO – Exosomes.
Figure 4.
Figure 4.
Distribution of miRNA among the different samples. Venn diagram analysis was used to determine shared and uniquely identified miRNA in the different samples. All the miRNA identified by combined P-values were used to analyze the sharing of miRNA between cells, apoptotic bodies, microvesicles and exosomes. ABs – Apoptotic Bodies, MVs – Microvesicles, EXO – Exosomes.
Figure 5.
Figure 5.
Clustering of uniquely identified miRNA and their association with biological processes. Hierarchical clustering of identified miRNAs in cells, apoptotic bodies, microvesicles and exosomes. The clusters are assembled together in 4 groups. The miRNA are indicated on the right and samples are indicated below. Group 1 represent Exosomes specific miRNA, group 2 – cell specific miRNA, group 3 – cell, apoptotic bodies, microvesicles shared miRNA and group 4 – cell and apoptotic bodies shared miRNA.
Figure 6.
Figure 6.
Comparative analysis of exosome specific miRNA and miRNA identified previously in cell lines and melanoma tissues and biopsies: Comparison of unique MML-1 exosomal microRNAs with databases of melanoma tissue and benign naevi microRNAs. (A) Heatmap showing the significant expression of hsa-miR-142-3p, hsa-miR-150, hsa-miR-155, hsa-miR-223, and hsa-miR-486-5p in melanoma biopsy as compared to the Nevi Biopsy. (B) Heatmap showing the significant expression of hsa-miR-142-3p, hsa-miR-335, hsa-miR-155, and hsa-miR-494 in melanoma tissue and melanoma cell lines as compared to benign naevus. This data was accessed from GEO database with accession number A) GSE34460 and B) GSE35579. An integrative statistical hypothesis testing method was applied for analyzing the P values of the statistically significant candidates. P < 0.05 was considered as statistically significant.

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