Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells – Evidence of unique microRNA cargos

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.

BRAFV600inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells

“…S1). However, the peaks for the rRNA subunits were not as prominent in the exosomes, supporting our previous findings arguing that smaller exosomes carry relatively little rRNA (5,13). The total RNA and small RNA profiles did not show a significant difference in the extracellular vesicle subset after treatment (Fig.…”

“…The melanoma cell lines MML-1 and A375, both having a BRAF V600E mutation, were cultured as described previously (5). Vemurafenib (PLX4032) and dabrafenib were dissolved in DMSO as recommended by the manufacturer instructions (Selleckchem).…”

“…The small RNA deep sequencing for the nontreated samples has previously been analyzed and published (5), and the same raw data were now reanalyzed together with the treated samples to determine the differences in the cells and extracellular vesicle subsets upon vemurafenib treatment. Analysis of the small RNA deep sequencing was focused on ncRNAs, and first an average of the duplicates of all of the samples was calculated and then the percentage of sequencing reads for the different RNA species was determined.…”

“…Small ncRNAs are 20-200 nucleotides (nt) in length and include species such as miRNAs, piRNAs, siRNAs, tRNAs, snRNAs, snoRNAs, vaultRNAs, and other less well-characterized RNA species (5). The functional role of these small RNAs, especially miRNA, siRNA, and piRNA, is gene silencing by interaction with chromatin or by base pairing with complementary mRNAs or DNAs (6)(7)(8)(9).…”

“…It has also been shown that extracellular vesicles can transfer functional RNA between cells (12). In addition, different subsets of vesicles such as apoptotic bodies, microvesicles, and exosomes contain distinct RNA molecules, especially miRNA, that are unique to different exosomal subsets (5,13). These observations have opened a field of research aiming to understand the vesicular contents and function under different conditions and how they influence the function of the vesicles.…”