Fig 2: The circLDLR-mediated ceRNA network. The ceRNA network of circLDLR (has_circ_0006877) was computationally constructed to predict the most likely sponged miRNAs and their affected targets. In the network, (1) the most likely putative miRNAs (top 6) with MREs on hsa_circ_0006877 were selected, including has-miR-1294, hsa-miR-1827, hsa-miR-326, hsa-miR-330-5p, hsa-miR-486-3p, and hsa-miR-649; (2) the target genes of putative miRNAs, which were also involved in ovarian steroidogenesis pathway, were selected, including CYP19A1, BMP6, INSR, AKRIC2, ADCY7, FZD4, PTPN11, and BZRAP1. Hsa_circ_0006877 was indicated in red; miRNAs in blue; and mRNAs in orange.

Fig 3: Confirmation of the direct role of circLDLR in miR-1294/ CYP19A1 axis. The miR-1294 mimics was transfected into the recipient KGN cells which has been treated with OE- circLDLR exosomes. 48- h later, the expressions of miR-1294 (A), CYP19A1 mRNA (B) and CYP19A1 protein (D) in the corresponding recipient cells were quantified by qRT-PCR and Western blot. At the same time, the estradiol secretions by the recipient cells were also measured by ELISA assay (C). GAPDH was used as the internal controls for circLDLR and CYP19A1. U6 was used as the internal controls for miR-1294. Each experiment was performed six times. exo-OE-circLDLR, exosomes with over-expressed circLDLR; exo-OE-NC, negative control exosomes. **** indicates p < 0.0001, ** indicates p < 0.01, NS, no significant difference. The results are presented as means ±SEM.

Fig 4: Validation of exosomal circLDLR regulation on CYP19A1 via miR-1294 by luciferase activity assay. The relative luciferase activity was measured following transfection of the wild-type or mutant CYP19A1 3’-UTR in recipient KGN cells after treated with (A) over-expressed circLDLR exosome (exo-OE-circLDLR) or (B) knocked down circLDLR exosome (exo-si-circLDLR), to confirm that exosomal transfer of circLDLR is truly regulatory on CYP19A1 expression via its miR-1294 binding site. The firefly luciferase activities were normalized by renilla luciferase activities. The experiments were performed independently in triplicates. ** indicates p < 0.01, NS indicates no significant difference. The results are presented as means ±SEM.

Fig 5: Transcriptional dysregulation of circLDLR-miR-1294-CYP19A1 ceRNA network in granulosa cells of PCOS. The abundance of circLDLR (A), miR-1294 (B) and CYP19A1 (C) were compared between PCOS and non-PCOS control by qRT-PCR. (D) Expression correlation between circLDLR and CYP19A1. GAPDH was used as the internal control for circLDLR and CYP19A1, while U6 was used as the internal control for miR-1294. N=25 vs 25. The results were presented as mean ± SEM.