Circulating exosome-circRNAs mediated downregulation of FGF9 through ceRNA mechanism aggravates renal fibrosis in diabetic nephropathy

Figures

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  Fig 1. Graphic abstract of the study.

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  Fig 2. Expression of serum circRNAs in patients with DN. (A) Representative images of exosome morphology by transmission electron microscopy. (B) The vesicle size distribution of a representative exosome was measured by nanoparticle tracking analysis (NTA). (C) CircRNA length distribution statistics. (D) CircRNA source distribution diagram. (E) CircRNA chromosome distribution. (F) Sample correlation heat map. (G) Principal component analysis (PCA) diagram. (H) Volcanic plot of different circRNAs. (I) Heat map of differential circRNAs expression profiles. (J) The first 8 meaningful differences presented by circRNAs.

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  Fig 3. Biological functional enrichment of differentially derived circRNA genes. (A) GO Bubble Chart enrichment analysis of differentially circRNAs derived genes. (B) KEGG enrichment analysis of differentially derived circRNAs genes. (C) Reactome enrichment analysis of differentially derived circRNAs genes.

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  Fig 4. Identification of differential expression analysis. (A) Boxplot of the combined data of the GSE30528 database and the GSE30529 database sample after batch-effect removal. (B) PCA diagram of GSE30528 database and GSE30529 database after data merger. (C) Volcanic plot of differential genes. (D) Heat map of the expression profiles of the first 10 up-regulated and down-regulated genes were taken.

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  Fig 5. Weighted Correlation Network Analysis to identify DN related feature genes. (A) The optimal soft threshold was determined to be 12. (B) Hierarchical clustering tree shows different modules related to DN. (C) Heat map of correlation between characteristic genes of different modules and DN. (D) Scatter plot of correlation between Gene and Module Membership (MM) and Gene Significance (GS) in pink module. (E) Scatter plot of correlation between turquoise module MM and GS. (F) Scatter plot of correlation between black module membership and DN gene traits.

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  Fig 6. Central gene screening and functional enrichment analysis. (A) Venn diagram of intersection of DEGs and characteristic genes. (B) Protein Interaction (PPI) networks of 34 differentially expressed proteins. (C) GO Bubble Chart of differentially expressed genes. (D) KEGG enrichment of differentially expressed genes. (E) Network regulatory diagram of FGF9 target miRNAs predicted by Network Analyst.

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  Fig 7. Construction of circRNA-miRNA-mRNA regulatory network. (A) The top ten miRNAs were displayed in order of Max-Score from higher to lower for differentially expressed circRNAs and miRNAs binding sites. (B) Regulatory network diagram of differentially expressed circRNAs and their predicted miRNAs. (C) Venn diagram of common miRNAs predicted by Network Analyst and miRanda and psRobot. (D) CeRNA regulatory network diagram of the four common miRNAs and their corresponding serum exosomes circRNAs and FGF9.

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  Fig 8. Validation of FGF9 expression in external database and NRK-52E cells. (A) The expression of FGF9 in the GSE30122 database was significantly decreased. (B) The expression of FGF9 in the GSE96804 database was significantly decreased. (C) AUC value of FGF9 in the GSE30122 database. (D) The AUC value of FGF9 in the GSE96804 database. (E) Representative western blots showed that FGF9 expression of NRK-52E under HG stimulation. (F) Summarized data of western blots showed the FGF9 expression was downregulated under HG stimulation. (G) Representative western blots showed that α-SMA protein expression after FGF9 treatment. (H) Summarized data of western blots showed that α-SMA protein expression was decreased after FGF9 treatment. (I) Representative western blots showed that vimentin protein expression after FGF9 treatment. (J) Summarized data of western blots showed that vimentin protein expression was decreased after FGF9 treatment. (K) Cell viability was measured by CCK8. (L) Representative images of immunofluorescence staining for a-SMA (green) and DAPI (blue) (Scale bars: 75 μm). * p < 0.05, ** p < 0.01, *** p < 0.001.