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

OBJECTIVE

To understand the pathogenesis of polycystic ovary syndrome (PCOS), investigating the molecular mechanism of granulosa cell dysfunction is vital.

DESIGN

On the basis of bioinformatics analysis, we analyzed the differentially expressed genes and their enrichment pathways between patients with PCOS and normal samples. We also analyzed the levels of CD14 and miR-296-3p in the patients with PCOS. Pearson correlation coefficient was used to analyze the correlation between CD14 expression and PCOS-related inflammatory factors. The CCK-8 assay, flow cytometry, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and western blotting were performed to explore the mechanism of CD14 modulation via miR-296-3p in KGN cells in vitro.

SUBJECTS: The gene expression data of patients with PCOS and healthy individuals in the gene expression omnibus (GEO) database, as well as the experimental data in KGN cells.

EXPOSURE: The KGN cells were subjected to the following interventions: transfection with miR-296-3p inhibitor to suppress miR-296-3p expression; transfection with small interfering ribonucleic acid (siRNA) 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 nuclear factor kappa-B (NF-κB) pathway.

RESULTS

CD14 was up-regulated in patients with PCOS 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. The 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 cotransfected 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 the NF-κB signaling pathway. Our findings contribute to further understanding of the pathogenesis of PCOS and provide potential biomarkers for PCOS.