Among the 188 miRNAs investigated, we identified 4 miRNAs, whose levels were significantly higher in LNCaP supernatant after US treatment compared to those detected in basal conditions: miR-425-5p (FC: 5.129,
p = 0.028), miR-365b-3p (FC: 4.698,
p = 0.036), miR-629-5p (FC: 2.274,
p = 0.038), and miR-193b-3p (FC: 3.034,
p = 0.018). Three of them, miR-425-5p, miR-365a-3p, and miR-193b-3p, were already described in the literature to be involved in PCa. In fact, miR-425-5p has been described to be overexpressed in PCa cell lines, where it promotes proliferation, migration, and invasion by targeting forkhead box J3 [
27]; miR-365b-3p expression resulted to be expressed at statistically different levels in PCa tissues compared to tissue from patients with prostatic hyperplasia [
28]; the silencing of miR-193b-3p through promoter methylation was shown to correlate with more aggressive PCa [
29]. Interestingly, we also identified miR-629-5p, which to our knowledge has never been linked to PCa disease (
Figure 2A,
Supplementary Table S1). Notably, two additional miRNAs, whose supernatant levels were increased more than two-fold following LNCaP US treatment—miR-374a-5p (FC: 2.550,
p = 0.060) and miR-194-5p (FC: 4.416,
p = 0.195)—were not described in the literature as PCa-related miRNAs. DU145 profiling showed a significant increase in the release of let-7d-5p (FC:2.694,
p = 0.007) following US treatment and, although this miRNA family has been related to several cancers [
30], no specific roles in PCa have been described for it (
Figure 2B,
Supplementary Table S1). We investigated the cellular release kinetics of the newly identified miR-629-5p, miR-374a-5p, miR-194-5p, and let-7d-5p by single RT-qPCR assay. The release of miRNAs in the supernatant of LNCaP and DU145 cells was measured after treating the cells with US for 15 min, 30 min, and 1 h. The supernatants of untreated control cells were collected before sonication, following incubation with exosomes-depleted medium for the same time. We observed a significant increase in the release of miR-629-5p, miR-374a-5p, and miR-194-5p in LNCaP supernatant after 1h of US treatment compared to untreated cells (miR-629-5p, FC: 6.5; miR-374a-5p, FC: 7.1; miR-194-5p, FC: 6.4), while no significant increase was observed at earlier time points (
Figure 3A–C).
Instead, we observed a time-dependent increase of let-7d-5p release in the supernatant from US-treated DU145 cells compared to untreated cells. This increase in miRNA release was already significant after 15 min of US treatment (FC: 2.8) and augmented when the cells were treated for longer times (30′ treatment, FC: 40; 1h treatment, FC: 81.3) (Figure 3D).