piR-823 and piR-20326 Differentially Regulate Stemness Programs in Ovarian Cancer Stem-like Cells

Ovarian cancer stem-like cells (OCSCs) are major drivers of tumor recurrence and chemoresistance. Although PIWI-interacting RNAs (piRNAs) have emerged as important regulators of cancer stem cell (CSC) biology, their roles in OCSCs remain poorly understood. We performed a systematic analysis of public datasets to identify ovarian cancer-associated piRNAs and selected piR-823 and piR-20326 for functional validation. Their expression was evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR)following piRNA mimic and inhibitor transfections in three-dimensional (3D) ovarian cancer spheroids and ALDH+/CD133+ stem-like subpopulations isolated by fluorescence-activated cell sorting. Stemness-associated genes (ALDH1A1, ALDH1A2, ALDH1A3, SOX2, CD133, and NANOG) were analyzed, and The Cancer Genome Atlas (TCGA) transcriptomic data were used for clinical validation. Both piR-823 and piR-20326 were significantly enriched in spheroid cultures and ALDH+/CD133+ populations, supporting their association with stem-like phenotypes. Functional analyses showed that piR-823 modulation was associated with changes in multiple stemness-related markers, whereas piR-20326 showed variable effects across two-dimensional (2D) and spheroid 3D culture models. TCGA data confirmed consistent co-expression patterns among stemness-related genes in clinical samples. These findings identify piR-823 as a key regulator and piR-20326 as a modulatory component of the piRNA-mediated stemness network in ovarian cancer, supporting further investigation of the piRNA–stemness axis as a potential therapeutic target.