Since US treatment showed to be effective in increasing the concentration of known miRNAs released in the supernatant of LNCaP and DU145 cells, we profiled the miRNAs released by these cells before and after US treatment, with the aim of identifying new potential PCa-related miRNA biomarkers. The miRNA profile analysis was performed by TaqMan™ Advanced miRNA Human Serum/Plasma RT-qPCR array cards. The volcano plots in Figure 2 show the log2FC of all miRNAs detected in LNCaP (Figure 2A) or DU145 (Figure 2B) supernatants after 1 h of US treatment compared to those released from untreated cells over the same time (see also Supplementary Table S1).
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 
; miR-365b-3p expression resulted to be expressed at statistically different levels in PCa tissues compared to tissue from patients with prostatic hyperplasia 
; the silencing of miR-193b-3p through promoter methylation was shown to correlate with more aggressive PCa 
. Interestingly, we also identified miR-629-5p, which to our knowledge has never been linked to PCa disease (Figure 2
A, 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 
, no specific roles in PCa have been described for it (Figure 2
B, 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 3
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).