The effect and regulation mechanism of CircHIAT1 on polycystic ovary syndrome

Abstract

Part I: Differential Expression of CircRNA in Polycystic Ovary Syndrome (PCOS) Objective: Circular RNA (circRNA) has been found to play an important role in many diseases, however, there has been limited research focusing on the circRNA in PCOS disease. Therefore, we intended to detect the expression of circRNAs in cumulus granulosa cells (CCs) of PCOS patients by high-throughput sequencing, to explore the role and possible mechanism of circRNAs in the pathogenesis of PCOS. Methods: The CCs from IVF/ICSI patients were collected, randomly. We selected 3 pairs of samples for high-throughput sequencing. 14 differentially expressed circRNAs were selected for clinical validation in CCs. Results: A total of 55,776 differentially expressed circRNAs was detected. Compared with the control group, 7,047 circRNAs were significantly up-regulated and 48,729 were significantly down-regulated in PCOS group. GO analysis showed circRNAs might be active in cell metabolism, biological regulation and active catalysis of PCOS. KEGG pathway analysis indicated that circRNAs could participate in signal transduction, substance transport and catabolism, endocytosis and ubiquitin mediated proteolysis pathway. The expression of circ_0011693, circ_0042454, circ_0001365, circ_0006252 and circ_0003737 were significantly higher in PCOS patients, while the expression of circHIAT1 was significantly lower in PCOS patients. Conclusions: The study found the differential expression profiles of circRNA in PCOS for the first time. Bioinformatics analysis showed that differentially expressed circRNAs could participate in multiple pathways related to PCOS diseases. Therefore, the effect and regulatory of circRNA on PCOS deserve further study. Part II: The Effects of CircHIAT1 on steroidogenesis in polycystic ovary syndrome by sponging miR-195-5p Objective: We hypothesised circHIAT1 could regulate the function of CCs by sponging miR-195-5p, thus participating in the regulation of PCOS. Methods: The study detected the co-localization of circHIAT1 and miR-195-5p in CCs by RNA-FISH. qRT-PCR and Western Blot were used to detect the effects of circHIAT1 on AR, CYP19A, GATA4, and VEGF in DHT pretreated KGN cell line. FACS was used to detect the cell cycle. The targeting relationship of circHIAT1/miR-195-5p, and miR-195-5p/CCNE1 were detected by dual-luciferase reporter assay. The expression of miR-195-5p and CCNE1 in clinical samples was detected. The effects of miR-195-5p and CCNE1 on AR, CYP19A, GATA4, VEGF, and cell cycle were verified. Results: circHIAT1 and miR-195-5p were both localized in the cytoplasm of primary CCs and KGN cells. circHIAT1 silencing could significantly increase the levels of AR, CYP19A, GATA4, and VEGF, and lead to the decrease of cell viability in KGN cells. circHIAT1 could bind to miR-195-5p. miR-195-5p was found highly expressed in CCs of PCOS patients. Overexpression of miR-195-5p could significantly increased the levels of AR, CYP19A, GATA4, and VEGF in KGN cells, and resulted in a significant decrease of cell viability. miR-195-5p could bind to CCNE1. The expression level of CCNE1 in PCOS group was significantly lower. Overexpression of CCNE1 could significantly decreased the levels of AR, CYP19A, GATA4 and VEGF, and increased the cell viability in KGN cells. miR-195-5p mimic could inhibit the regulation of circHIAT1 pEX-3 on AR, CYP19A, GATA4 and VEGF. Conclusion: Silencing circHIAT1 could significantly increase steroidogenesis related indicators, inhibit the cell cycle of KGN cells. Thus, circHIAT1 might be used as a new target for the diagnosis and treatment of PCOS.