Plasma-derived extracellular vesicles miR-335-5p as potential diagnostic biomarkers for fusion-positive rhabdomyosarcoma

Abstract

Background: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, with embryonal (ERMS) and alveolar (ARMS) representing the two most common histological subtypes. ARMS shows poor prognosis, being often metastatic at diagnosis. Thus, the discovery of novel biomarkers predictive of tumor aggressiveness represents one of the most important challenges to overcome and may help the development of tailored therapies. In the last years, miRNAs carried in extracellular vesicles (EVs), small vesicles of endocytic origin, have emerged as ideal candidate biomarkers due to their stability in plasma and their tissue specificity.

Methods: EVs miRNAs were isolated from plasma of 21 patients affected by RMS and 13 healthy childrens (HC). We performed a miRNA profile using the Serum/Plasma Focus microRNA PCR panels (Qiagen), and RT-qPCR for validation analysis. Statistically significant (p < 0.05) miRNAs were obtained by ANOVA test.

Results: We identified nine EVs miRNAs (miR-483-5p, miR-132-3p, miR-766-3p, miR-454-3p miR-197-3p, miR-335-3p, miR-17-5p, miR-486-5p and miR-484) highly upregulated in RMS patients compared to HCs. Interestingly, 4 miRNAs (miR-335-5p, miR-17-5p, miR-486-5p and miR-484) were significantly upregulated in ARMS samples compared to ERMS. In the validation analysis performed in a larger group of patients only three miRNAs (miR-483-5p, miR-335-5p and miR-484) were differentially significantly expressed in RMS patients compared to HC. Among these, mir-335-5p was significant also when compared ARMS to ERMS patients. MiR-335-5p was upregulated in RMS tumor tissues respect to normal tissues (p = 0.00202) and upregulated significantly between ARMS and ERMS (p = 0.04). Furthermore, the miRNA expression correlated with the Intergroup Rhabdomyosarcoma Study (IRS) grouping system, (p = 0.0234), and survival (OS, p = 0.044; PFS, p = 0.025). By performing in situ hybridization, we observed that miR-335-5p signal was exclusively in the cytoplasm of cancer cells.

Conclusion: We identified miR-335-5p as significantly upregulated in plasma derived EVs and tumor tissue of patients affected by ARMS. Its expression correlates to stage and survival in patients. Future studies are needed to validate miR-335-5p as prognostic biomarker and to deeply elucidate its biological role.

Keywords: Extracellular vesicles; Liquid biopsy; MicroRNA; Rhabdomyosarcoma.

Fig. 1

Heatmap of the 41 statistically dysregulated EVs-miRNAs in ERMS and ARMS patients compared to HC. Upregulated and downregulated miRNAs are represented in red and green, respectively, P-value < 0.05

Fig. 2

Significant EVs-miRNAs expression in the validation analysis evaluated by RT-qPCR. ** p < 0,01 (miRNA-484, miRNA-483-5p, miRNA-335-5p)

Fig. 3

Bioinformatic analysis of predicted targets of miR-335-5p. A GO enrichment analysis; B KEGG Pathways; C Cytoscape interaction network

Fig. 4

Bioinformatics analysis of expression of miR-335-5p, from GSE135518 database in RMS tissues compared to normal tissue. A Comparison between miR-335-5p expression and histology B Comparison between miR-335-5p and IRS; C Correlation between miR-335-5p and OS and (D) PFS**** p < 0,0001; ** p < 0,01; * p < 0,05

Fig. 5

Representative miR-335-5p ISH staining images in tissue samples of RMS patients (× 400). The panel shows the presence of miR-335-5p in tumor cells