Bioinformatics analysis and experimental validation of potential targets and pathways in chronic kidney disease associated with renal fibrosis.

BACKGROUND

Chronic kidney disease (CKD) has emerged as a major health problem worldwide. Previous studies have shown that specific miRNA expression profiles of patients with CKD are significantly changed. In this study, we aim to elucidate the role of miRNAs as potential biomarkers in CKD progression by integrating bioinformatics analysis with experimental validation, thereby providing medical evidence for the prevention and treatment of CKD.

METHOD

Bioinformatics analysis was used to identify potential targets and pathways in CKD-associated renal fibrosis through randomly obtaining miRNA microarray data related to CKD patients in the Gene Expression Omnibus (GEO) database according to the inclusion and exclusion criteria, conducting pathway enrichment analysis and constructing protein-protein interaction (PPI) networks and miRNA-mRNA network by Cytoscape 3.8.0. In vitro experiments were employed to verify the role and mechanism of miR-223-3p in human renal tubular epithelial cells (HK2) through Quantitative real-time PCR assays, Western blot, Immunofluorescence analysis and Double luciferase reporter gene experiment. Multi-group one-way analysis of variance (ANOVA) and the Dunnett-t test were uesd to analyze the results by SPSS24.0.

RESULTS

10 up-regulated and 11 down-regulated miRNAs of CKD patients were screened out. Phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) was the first pathway of pathway enrichment analysis. MiR-223-3p (logFC=-2.047, p = 0.002) was one of the four hub miRNAs. Furthermore, we observed a reduction in α-smooth muscle actin (α-SMA) (p = 0.001) and Collagen type I alpha 1 (Col1-a1) (p = 0.023) levels upon miR-223-3p overexpression, which aligned with our bioinformatics predictions. This downregulation was attributed to the inhibition of nuclear factor kappa-B (NF-κB) nuclear translocation and subsequent decrease in the secretion of inflammatory cytokines, such as interleukin-6 (IL-6) (p = 0.005). Conversely, when CHUK was further overexpressed, the inhibitory effect of miR-223-3p on epithelial-mesenchymal transition (EMT) was attenuated, confirming the specific interaction between miR-223-3p and CHUK.

CONCLUSION

Our findings provide compelling evidence that miR-223-3p acts as a suppressor of EMT in CKD by specifically targeting the CHUK and modulating the PI3K/Akt pathway, which holds great promise as a novel therapeutic target for CKD treatment. Additionally, this study offers a potential avenue for the development of future interventions aimed at halting or reversing the progression of CKD.