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Emerging studies demonstrate that PIWI-interacting RNAs (piRNAs) are associated with various human cancers. This study aimed to evaluate the urinary extracellular vesicles (EVs) piRNAs as non-invasive biomarkers for prostate cancer (PCa) diagnosis. RNA was extracted from urinary EVs from five PCa patients and five healthy controls (HC), and the piRNAs were analyzed by small RNA sequencing. Dysregulated piRNAs were identified and then validated in another 30 PCa patients and 10 HC by reverse-transcription polymerase chain reaction (RT-qPCR). The expressions of novel_pir349843, novel_pir382289, novel_pir158533, and hsa_piR_002468 in urinary EVs were significantly increased in the PCa group compared with the HC group. The area under the curve (AUC) of novel_pir158533, novel_pir349843, novel_pir382289, hsa_piR_002468, and the combination of the four piRNA in PCa diagnosis was 0.723, 0.757, 0.777, 0.783, and 0.853, respectively. After the RNAhybrid program analysis, all four piRNAs had multiple potential binding sites with key mRNAs in PTEN/PI3K/Akt, Wnt/beta-catenin, or androgen receptor pathway, which are critical in PCa development and progression. In conclusion, our findings indicate that specific piRNAs in urinary EVs may serve as non-invasive diagnostic biomarkers for PCa.

Identification of piRNA Targets in Urinary Extracellular Vesicles for the Diagnosis of Prostate Cancer