TP53 mutations correlate with the non-coding RNA content of small extracellular vesicles in melanoma

Abstract

Non-coding RNAs (ncRNAs) are important regulators of gene expression. They are expressed not only in cells, but also in cell-derived extracellular vesicles (EVs). The mechanisms controlling their loading and sorting remain poorly understood. Here, we investigated the impact of TP53 mutations on the non-coding RNA content of small melanoma EVs. After purification of small EVs from six different patient-derived melanoma cell lines, we characterized them by small RNA sequencing and lncRNA microarray analysis. We found that TP53 mutations are associated with a specific micro and long non-coding RNA content in small EVs. Then, we showed that long and small non-coding RNAs enriched in TP53 mutant small EVs share a common sequence motif, highly similar to the RNA-binding motif of Sam68, a protein interacting with hnRNP proteins. This protein thus may be an interesting partner of p53, involved in the expression and loading of the ncRNAs. To conclude, our data support the existence of cellular mechanisms associate with TP53 mutations which control the ncRNA content of small EVs in melanoma.

Keywords: Small extracellular vesicle; TP53 mutations; long non‐coding RNA; microRNA.

FIGURE 1

Examples of characterization of small EVs purified from a WT TP53 cell line (M113) and a mutant TP53 cell line (M117). (a) Representative transmission electron microscopy (TEM) images of melanoma‐derived small EVs. Circular morphology and the absence of internal staining indicate intact, compartmentalized vesicles. (b) Size distributions of the three independent small EV purifications/productions by cell line by qNano technology. Size is consistent with results from TEM. (c) Protein characteristics of small EVs using western blotting technique. For consistency of comparison between small EVs and cells, 3 μg of sample was loaded per well for western blotting. ***p < 0.001.

FIGURE 2

Differential miRNA content of small EVs from TP53‐mutated or WT melanoma cells. (a) Volcano plot of adjusted p value as a function of weighted fold‐change for small RNA (adjusted p value < 0.05). Red dots on the left represent significantly downregulated small RNAs in small EVs from TP53 WT melanoma cells and on the right the upregulated ones compared to small EVs from mutant TP53melanoma cells. In green, miRNAs with a log2 fold change >2 or −2. (b) Enrichment analysis of the 1888 targets of the significantly enriched miRNAs in small EVs derived from TP53 mutant cells. (c) Enrichment analysis of the 496 targets of the significantly enriched miRNAs in small EVs derived from TP53 WT cells. (d) Volcano plot of differentially expressed small RNA between small EVs from NRAS or BRAF mutated melanoma cell lines. (e) Expression level of hsa‐miR‐155‐5p in small EVs from NRAS and BRAF mutated melanoma cells. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Sam68 is overexpressed in the nucleus of mutant TP53 melanoma cell lines to regulate RNA expression. (a) 8‐mer motif enriched in RNA overexpressed in mutant TP53 small EVs. On the top, RNA motif found in our enriched RNA according to MEME. On the bottom, the RNA‐binding motif of KHDRBS1 protein (Sam68) found through Tomtom analysis. (b) Quantification of Sam68 expression and differences between TP53 WT and TP53 mutant melanoma cells by western blot analysis. The optical density of each sample was measured and normalized using a housekeeping protein (Actin) run on the same gel using ImageJ. Data are expressed as relative expression (ratio Sam68/Actin), n = 3. (c) Measurement of Sam68 expression in the cytoplasm, the nucleus and the small EVs of TP53 mutant (M117) and WT (M113) melanoma cell lines by western blot analysis. Normalization of the Sam68 protein input was performed using the actin protein in the cytoplasm and in small EVs and using the Histone 3 protein in the nucleus. *p < 0.05.

FIGURE 4

Intronic miRNA expressions are correlated with Sam68 expression in TP53 mutated melanoma patients from TCGA. (a) Expression in melanoma cells lines of the miRNAs overexpressed in TP53 mutant small EVs and of one downregulated (hsamiR‐ 5582‐3p) (WT: M113, M28, M6 and mut: M117, M18, M88), n = 3. (b) Sam68 expression level in TP53 mutated cell line M117 after transfection with a siRNA targeting Sam68 or with a scramble siRNA (si Neg). Normalization of the Sam68 protein input was performed using the actin protein. (c) Relative Sam68 mRNA expression was determined by qPCR after normalization with the RPLP0 housekeeping gene, n = 3 (d) Expression of our candidate miRNAs overexpressed in TP53 mutant small EVs in M117 cells transfected with Sam68 siRNA compared to siNeg, n = 3. (e) Western blot probed with anti‐Sam68 antibody to assess Sam68 associated with the captured beads following Sam68 immunoprecipitation compared to control IgG immunoprecipitated and total Input. (f) PCR analysis of candidate miRNAs following immunoprecipitation of Sam68. *p < 0.05, **p < 0.01, ***p < 0.001.