The Association of Cell-Free LncH19 and miR-29b Expression with the PI3K/AKT/HIF-1/VEGF Pathway in Patients with Diabetic Nephropathy: In Silico Prediction and Clinical Validation

Figures

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  Figure 1. A flow chart of in silico data analysis for the miRNA target prediction enrichment pathway analysis and miRNA–disease interaction.

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  Figure 2. miR-29–lncH19 pairing showing the target binding sites. Source: starBase v2. database (http://starbase.sysu.edu.cn/, accessed 2 January 2024).

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  Figure 3. MicroRNA-29B and lncH19 gene structural analysis. (A) miRNA-29b chromosomal location and (B) miR-29b secondary structure, (C) lncH19 chromosomal location, and (D) lncH19 secondary structure. Source: https://useast.ensembl.org/Homo_sapiens/Gene/; last accessed 25 December 2024).

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  Figure 4. MiR-29b-3p, lncH19, and VEGF evidence of cross interactions. (A) miR-2b with VEGF; (B) lncH19 loop interaction with miR-29b and VEGF.

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  Figure 5. Interaction analysis for “PI3K/AKT/HIF-1” pathway. (A) Target pathways and genes (red boxes) in enriched KEGG pathway. The original pathway is adopted with permission (https://www.kegg.jp/pathway/map04066) (last accessed 20 November 2024) [23], (B) protein–protein interaction and clusters, and (C) coexpression analysis (Data sources: STRING database).

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  Figure 6. Gene expression among the four investigated groups, including the DM (green), DM with microalbuminuria (blue), and DM with macroalbuminuria (red) for lncH19 (panel A), miR-29b (panel B), their ratio (panel C) and target genes (i.e., panel D: PI3K, panel E: AKT, panel F: mTOR, and panel G: HIF-1). Data are presented as median and interquartile range; Kruskal–Wallis was employed to calculate the p-values, where * denotes a significant difference vs. the control group, # indicates a significant difference vs. the DM group, and $ indicates significant differences vs. the microalbuminuria group at p < 0.05.

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  Figure 7. VEGFA protein analysis. (A) Circulating levels of serum VEGFA in the four studied groups. (B) The correlation between eGFR and the serum level of VEGFA. (Data were presented as median and interquartile range. Kruskal–Wallis tests were used to calculate the p-value where * denotes a statistically significant difference vs. the control group, # indicates a significant difference vs. the diabetic group, and $ indicates a significant difference vs. the microalbuminuria group at p < 0.05. R2 indicates the results of the Spearman correlation test, and the significance was determined at p < 0.05.

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  Figure 8. An illustration of the correlation matrix showing the connection between lncH19 level and various study variables. The blue color represents positive correlations, while the red color represents negative correlations. The value of R represents the strength of the association. Statistical significance was determined if p-value < 0.05.

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  Figure 9. VEGF and lncH19 as an indicator for prognosis. ROC curve analysis for VEGF and lncH19 distinguishes patients with DN from those without DN. AUC: area under the curve, CI: Confidence Interval. * Statistical significance at p-value less than 0.05.