Supplemental Material for: Expression of miR-382-5p in polycystic ovary syndrome and its role in regulating granulosa cell function via targeting TOP1

The protein content of TOP1 in KGN cells was detected by Western blotting. RNA immunoprecipitation (RIP) and dual-luciferase assays were used to validate the binding relationship.</p><p dir="ltr">Results:</p><p dir="ltr">Serum miR-382-5p levels were significantly downregulated in PCOS patients, demonstrating good diagnostic value and showing a negative correlation with key clinical indicators of PCOS. In vitro experiments confirmed that, under insulin-induced conditions, overexpression of miR-382-5p significantly inhibited insulin-induced abnormal proliferation of granulosa cells, downregulated the protein of cyclin D1, and simultaneously promoted apoptosis by upregulating the pro-apoptotic protein Bax and downregulating the anti-apoptotic protein Bcl-2.

This mechanism was associated with the direct targeting and inhibition of topoisomerase I (TOP1). In PCOS patients, elevated TOP1 mRNA and protein expression correlated negatively with miR-382-5p levels. Overexpression of TOP1 significantly reversed the antiproliferative and proapoptotic effects of miR-382-5p.</p><p dir="ltr">Limitations:</p><p dir="ltr">The verification of the miR 382 5p/TOP1 regulatory mechanism was limited to incomplete in vitro cell experiments and non-standard clinical detection, which cannot fully confirm its exact binding effect, in vivo cascade specificity, and independent pathogenic causality in PCOS patients.

Additionally, all PCOS patients presented oligo-/anovulation, whereas healthy controls had regular menstrual cycles; given that circulating miR‑382‑5p levels vary with menstrual cycle phases, the observed downregulation in PCOS serum may partly stem from disrupted cyclic ovulation rather than representing an intrinsic PCOS-specific molecular alteration. Therefore, miR‑382‑5p should be interpreted as a correlative biomarker associated with PCOS phenotypes rather than an independent pathogenic mediator.</p><p dir="ltr">Conclusions:</p><p dir="ltr">miR-382-5p regulated the imbalance between insulin-mediated proliferation and apoptosis in KGN cells by directly targeting TOP1, thereby contributing to the progression of PCOS. </p>