Molecular characterization of extracellular vesicles derived from follicular fluid of women with and without PCOS: integrating analysis of differential miRNAs and proteins reveals vital molecules involving in PCOS

Figures

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  Fig. 1. Isolation and identification of FF-EVs. (A) The route and methods of this research. (B) TEM results of extracellular vesicles derived from FF of PCOS patients and healthy women. (C) NTA results profile of extracellular vesicles from ovarian FF of PCOS patients and healthy women. (D) Western blotting is used to verify isolated FF-EVs without granulosa contamination. CD63 and β-actin were used as primary antibodies, respectively. (E) The extracellular vesicles isolated from ovarian FF of PCOS patients and healthy women were verified by western blot analysis

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  Fig. 2. Comparison of expression levels of the up-regulated and down-regulated of top 20 DEmiRNAs in 6 FF-EVs samples. (A) Histogram of upregulated and downregulated of top 20 miRNAs. (B) Clustering analysis of upregulated and downregulated of top 20 miRNAs

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  Fig. 3. GO function annotation and KEGG pathways analysis of DEmiRNAs in FF-EVs. (A) The GO terms of 514 DEmiRNAs, including BP on the left, CC in the middle, and MF on the right. (B) Multi-class scatter plot of GO terms analysis of 514 DEmiRNAs. (C) Scatter plot of KEGG enrichment analysis of 514 DEmiRNAs. (D) The GO terms of 628 candidate predicted target genes of 6 DEmiRNAs, including BP on the left, CC in the middle, and MF on the right. (E) Multi-class scatter plot of GO terms analysis of 628 candidate predicted target genes of 6 DEmiRNAs. (F) Scatter plot of KEGG enrichment analysis of 628 candidate predicted target genes of 6 DEmiRNAs

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  Fig. 4. Ingenuity pathway analysis of DEmiRNAs in FF-EVs. Upregulated miRNAs are shown in red, and downregulated miRNAs are shown in green

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  Fig. 5. TMT assay validation and preliminary GO and KEGG analyses of 2487 DEPs in FF-EVs. (A) TMT assay validation by western blotting. (B and C) GO function annotation of 2487 DEPs. (D) Scatter plot of KEGG enrichment analysis of all quantifiable proteins

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  Fig. 6. Comparison of the expression levels of the highly enriched 32 DEPs in FF-EVs in 6 FF-EVs samples and their subcellular localization. (A) Heatmap and Clustering of 32 DEPs in FF-EVs. (B) Histogram of 32 DEPs in FF-EVs. (C) Subcellular localization of 32 DEPs in FF-EVs. (D and E) The GO terms of DEPs include BP, CC, and MF. (F) Scatter plot of KEGG enrichment analysis of 32 DEPs in FF-EVs

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  Fig. 7. Correlation diagram of DEmiRs target genes and DEPs. There was a corresponding relationship between the target gens of 6 differential miRNAs and DEPs. The red and green color represented upregulation and downregulation DEPs, respectively

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  Fig. 8. Possible molecular mechanisms of GRAMD1B and SPTLC2 involving in PCOS. (A) GRAMD1s comprise an N-terminal GRAM domain and a StART-like domain, which is followed by a C-terminal transmembrane domain that anchors the protein to the ER. GRAMD1s move to ER-PM (plasma membrane) contacts by sensing accessible PM cholesterol via the GRAM domain and can transport accessible cholesterol from the PM to the ER via the StART-like domain. GRAMD1s family are important for cholesterol homeostasis. GRAMD1B protein presented a downregulated level in FF-EVs of PCOS patients. Simultaneously, gramd1b gene is the predicted targets of 5 DEmiRNAs, including has-mir-3131, has-mir-206, has-mir-204-5p, has-mir-100-5p, and has-mir-193a-5p, found to be upregulated in FF-EVs of PCOS patients. An imagined work scenario should be decreased GRAMD1 expression or loss of GRAMD1 function caused by upregulated miRNAs led to sustained accumulation of accessible cholesterol in the PM and possible dysregulation of cellular cholesterol homeostasis as well as altered steroid hormone production and activity, eventually resulting in POCS. (B) SPT is a rate-limiting step in the de novo ceramide biosynthesis pathway and is…