The deficiency of CD14 weakens the biological function of miR-296-3p inhibitor in polycystic ovary syndrome granulosa cell by inhibiting NF-κB pathway.

Objective

Investigating the molecular mechanism of granulosa cell dysfunction is vital to understand the pathogenesis of polycystic ovary syndrome (PCOS).

Design

Based on bioinformatics analysis, we analyzed the differentially expressed genes and their enrichment pathways between PCOS patients and normal samples. We also analyzed the levels of CD14 and miR-296-3p in PCOS patients. Pearson correlation coefficient was used to analyze the correlation between CD14 expression and PCOS-related inflammatory factors. CCK-8 assay, flow cytometry, qRT-PCR and western blotting were performed to explore the mechanism of CD14 modulated via miR-296-3p in KGN cells in vitro. SUBJECTS The gene expression data of PCOS and normal individuals in the GEO database, as well as the experimental data in KGN cells. EXPOSURE KGN cells were subjected to the following interventions: Transfection with miR-296-3p inhibitor to suppress miR-296-3p expression; Transfection with small interfering RNA targeting CD14 (si-CD14) to knock down CD14; Co-transfection with si-CD14 and miR-296-3p inhibitor to evaluate the rescue effect. The detections included CCK-8, flow cytometry, qRT-PCR and Western blotting. MAIN OUTCOME MEASURES The expression of CD14 in patients with PCOS and KGN cells, and its correlation with inflammatory factors; The expression of MiR-296-3p in patients with PCOS, and its regulatory relationship with CD14; The effect of MiR-296-3p/CD14 on the viability and apoptosis of KGN cells; The influence of MiR-296-3p/CD14 on the NF-κB pathway.

Results

CD14 was up-regulated in PCOS patients and KGN cells, and the expression of CD14 was positively correlated with the expression of inflammatory factors (CAT, CCL2, ICAM1, IL10, MMP9 and TGFB1) related to PCOS. miR-296-3p was the upstream regulatory factor of CD14, and miR-296-3p inhibitor could promote cell apoptosis and inhibit cell viability. On the contrary, knockdown of CD14 in KGN cells inhibited apoptosis and promoted cell viability. Importantly, KGN cells co-transfected with si-CD14 and miR-296-3p could reverse the effects of miR-296-3p inhibitor and si-CD14 on the biological behavior of KGN cells. Moreover, miR-296-3p/CD14 axis mediated activation of NF-κB pathway in KGN cells.

Conclusions

Down-regulation of CD14 attenuated the miR-296-3p inhibitor-induced increase in apoptosis and decrease in cell viability by suppressing NF-κB signaling pathway. Our findings contribute to further understanding of the pathogenesis of PCOS and provide potential biomarkers for PCOS.