MicroRNA-371-373 cluster extracellular vesicle-based communication in testicular germ cell tumors

Abstract

Testicular germ cell tumors (TGCTs) represent the most common type of cancer in young adults. The cluster of microRNAs 371-373 is highly upregulated in TGCTs, and detection of miR-371a-3p specifically is currently being developed for clinical implementation as a sensitive and specific biomarker for TGCT, except for teratoma. Extracellular vesicles (EVs) are nano-sized particles used for cell communication, being increasingly regarded as potential sources of cancer biomarkers. Thus, the aim of this study was to characterize EVs from a wide range of TGCT samples, including cell lines, tissue explants and matched plasma samples from patients and healthy donors, and then use these samples to assess microRNA expression (miR-371-373 cluster and let-7e). TGCT-derived EVs were successfully isolated and characterized according to MISEV guidelines. TGCT cell lines showed different levels of EV-derived miR-371-373 cluster and let-7e. Upon differentiation of NT2 cells with ATRA, both cellular and EV-derived miR-371-373 cluster were downregulated, whereas let-7e was upregulated. TGCT patient samples presented high levels of EV-derived miR-371-373, except for the teratoma samples. We conclude that a significant portion of the circulating miR-371-373 cluster used as a TGCT biomarker in the clinic is secreted into EVs, and that this cluster and the let-7 family of microRNAs may be related with TGCT intercellular communication and differentiation.

Fig. 1

Secretion range (particles/cell) for the separate cell lines in lEV (A) and sEV (B); particle number/ml (C) and particle size (D) for both EV populations in all basal cell line experiments. Data shown as mean ± SEM for 3 independent experiments. ***—p < 0.001, ****—p < 0.0001

Fig. 2

EV characterization by western blot for traditional EV markers and cellular contaminants in representative cell line and tissue-derived EVs (A) and plasma-derived EVs (B), and TEM imaging (C) for TGCT cell lines (C1, C2 for TCam-2 lEV and sEV and C3,C4 for JEG-3 lEV and sEV), tissue (C5,C6) and plasma (C7,C8) EVs. Higher exposure time had to be used for plasma EV samples western blot (B) when compared with (A)

Fig. 3

MicroRNA levels for miR-371a-3p, miR-372-3p, miR-373-3p and let-7e in (T)GCT cell lines’ internal levels (A, D, G, J), and respective large (B, E, H, K) and small EVs (C, F, I, L). Data shown as mean ± SEM for 3 independent experiments

Fig. 4

Pluripotency and differentiation-related genes expression levels for NT2 cells treated with ATRA (A – NANOG, B – POU5F1, C – SOX2, D – PAX6, E – LIN28, F – LIN28B), compared with vehicle controls. Data shown as mean ± SEM for 5 independent experiments; *—p < 0.05, **—p < 0.01

Fig. 5

MiR-371–373 cluster (A, B, C) and let-7e (D) levels for NT2 cells treated with ATRA, compared with vehicle-treated controls. Data shown as mean ± SEM for 5 independent experiments; *—p < 0.05, **—p < 0.01

Fig. 6

MiR-371–373 cluster and let-7e EV-derived levels for NT2 cells treated with ATRA, compared with vehicle-treated controls, for both EV isolated populations (lEV in A-D, sEV in E–H). Data shown as mean ± SEM for 5 independent experiments; *—p < 0.05, **—p < 0.01

Fig. 7

Normalized number of particles secreted in TGCT tissue and adjacent testicular parenchyma, for lEV (A) and sEV (B). Data shown as mean ± SEM; *—p < 0.05, ***—p < 0.001

Fig. 8

EV-derived microRNA levels for miR-371a-3p (A, B), miR-372-3p (C, D), miR-373-3p (E, F) and let-7e (G, H), in non-TE TGCT, TE and adjacent testicular parenchyma tissue explants, for lEV and sEV. Data shown on a log scale as mean ± SEM; *—p < 0.05, **—p < 0.01, ***—p < 0.001, ****—p < 0.0001

Fig. 9

EV-derived microRNA levels (40—Ct) for miR-371a-3p (A, B), miR-372-3p (C, D), miR-373-3p (E, F) and let-7e (G, H), in non-TE TGCT, TE and HD plasma samples, for lEV and sEV. Data shown as mean ± SEM; *—p < 0.05