Follicular fluid-derived exosomal miR-143-3p/miR-155-5p regulate follicular dysplasia by modulating glycolysis in granulosa cells in polycystic ovary syndrome

Abstract

Objective: Polycystic ovary syndrome (PCOS) is characterized by follicular dysplasia. An insufficient glycolysis-derived energy supply of granulosa cells (GCs) is an important cause of follicular dysplasia in PCOS. Follicular fluid (FF) exosomal microRNAs (miRNAs) have been proven to regulate the function of GCs. In this study, exosomes extracted from clinical FF samples were used for transcriptome sequencing (RNA-seq) analysis, and a human ovarian granulocyte tumour cell line (KGN cells) was used for in vitro mechanistic studies.

Methods and results: In FF exosomal RNA-seq analysis, a decrease in glycolysis-related pathways was identified as an important feature of the PCOS group, and the differentially expressed miR-143-3p and miR-155-5p may be regulatory factors of glycolysis. By determining the effects of miR-143-3p and miR-155-5p on hexokinase (HK) 2, pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), pyruvate, lactate and apoptosis in KGN cells, we found that upregulated miR-143-3p expression in exosomes from the PCOS group inhibited glycolysis in KGN cells; knockdown of miR-143-3p significantly alleviated the decrease in glycolysis in KGN cells in PCOS. MiR-155-5p silencing attenuated glycolytic activation in KGN cells; overexpression of miR-155-5p significantly promoted glycolysis in KGN cells in PCOS. In this study, HK2 was found to be the mediator of miR-143-3p and miR-155-5p in FF-derived exosome-mediated regulation of glycolysis in KGN cells. Reduced glycolysis accelerated apoptosis of KGN cells, which mediated follicular dysplasia through ATP, lactate and apoptotic pathways.

Conclusions: In conclusion, these results indicate that miR-143-3p and miR-155-5p in FF-derived exosomes antagonistically regulate glycolytic-mediated follicular dysplasia of GCs in PCOS. Video Abstract.

Keywords: Exosomes; Glycolysis; HK2; Polycystic ovary syndrome; miR-143-3p; miR-155-5p.

Fig. 1

Characterization of FF-derived exosomes (A) representative western blotting of FF exosomal positive marker proteins (CD9, CD63) isolated from the control and PCOS groups. B Transmission electron microscopy (TEM) of FF exosomes isolated from the control group and PCOS group. C Representative nanoparticle tracking analysis (NTA) of exosomes isolated from the control and PCOS groups

Fig. 2

Exosomal RNA-seq data of the control group and PCOS group. C1–C6 were 6 random samples from the control group, and T1–T6 were 6 random samples from the PCOS group. A Heatmap showing that miRNAs were differentially expressed between the control and PCOS groups. B Heatmap showing hierarchical cluster analysis of mRNAs in the control and PCOS groups. C Volcano plot of differentially expressed miRNAs. D Volcano plot of differentially expressed mRNAs. E Correlation of expression changes observed by RNA-seq (Y-axis) and qPCR (X-axis)

Fig. 3

Enrichment analysis of differentially expressed genes in exosomal RNA-seq. A GO (Gene Ontology) analysis of differentially expressed miRNAs and mRNAs. The colour of the column indicates upregulation or downregulation, and the size of the dot indicates the number of genes, as shown on the right. B KEGG enrichment analysis of differentially expressed miRNAs and mRNAs. The colour and size of the dots represent − log10 (p value) and gene number, respectively, as shown on the right. C Heatmap of differentially expressed genes in the glycolytic pathway

Fig. 4

The interaction between miRNAs and the targeted genes. A–E Prediction targets between miR-143-3p/miR-155-5p and key glycolytic genes (HK2, PKM2, LDHA). F–J Luciferase reporter vectors. K–O Inhibitory rate of miRNA on luciferase activity of WT and MUT. mfe (hsa-miR-143-3p) = −43.8 kcal/mol, mfe (hsa-miR-155-5p) = −46.39 kcal/mol (*p < 0.05 and ***p < 0.001)

Fig. 5

The state of KGN cells with PCOS environment changes. A The expression of miR-143-3p and miR-155-5p in exosomes was analysed by RNA-seq. B The expression of exosomal miR-143-3p and miR-155-5p was analysed by qPCR. C The expression of miR-143-3p and miR-155-5p in KGN cells was analysed by qPCR. D The expression of glycolytic DEGs (HK2, PKM2 and LDHA) in KGN cells was analysed by qPCR. E Concentration of glycolytic products in KGN cell culture medium. F Culture medium characterization of KGN cells. G The Bcl2/Bax expression ratio was analysed by qPCR. H EDU shows the cell proliferation index (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 6

The expression changes of miR-143-3p can affect the glycolysis and proliferation of KGN cells. A qPCR analysis of miR-143-3p expression in cell models. B qPCR analysis of the expression of key DEGs (HK2, PKM2, LDHA) in cell models in a control environment. C qPCR analysis of the expression of key DEGs (HK2, PKM2, LDHA) in cell models in a PCOS environment. D Western blot analysis of HK2 in cell models. E Western blot analysis of PKM2 in cell models. F Western blot analysis of LDHA in cell models. G Metabolic concentration of pyruvate in cell model medium. H Metabolic concentration of lactate in cell model medium. I Culture medium characterization of the cell model. J qPCR analysis of Bcl2/Bax in cell models. K Western blot analysis of Bcl2/Bax in cell models. L EDU shows the proliferation index of the cell model (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 7

The expression changes of miR-155-5p can affect the glycolysis and proliferation of KGN cells. A qPCR analysis of miR-155-5p expression in cell models. B qPCR analysis of the expression of key DEGs (HK2, PKM2, LDHA) in cell models in a control environment. C qPCR analysis of the expression of key DEGs (HK2, PKM2, LDHA) in cell models in a PCOS environment. D Western blot analysis of HK2 in cell models. E Western blot analysis of PKM2 in cell models. F Western blot analysis of LDHA in cell models. G Metabolic concentration of pyruvate in cell model medium. H Metabolic concentration of lactate in cell model medium. I Culture medium characterization of the cell model. J qPCR analysis of Bcl2/Bax in cell models. K Western blot analysis of Bcl2/Bax in cell models. L EdU shows the proliferation index of the cell model (ns = not significant, *p < 0.05, **p < 0.01 and ***p < 0.001)

Fig. 8

FF-derived exosomal miR-143-3p/miR-155-5p mediate follicular dysplasia by antagonizing glycolysis of GCs in PCOS