Regulatory role of the circHIPK3/miR-125a-5p/NDUFA5 axis in diabetic retinopathy: combined analysis of single-cell and bulk transcriptomes.

Abstract

Objective: To explore the expression localization, functional effects, and upstream regulatory mechanism mediated by circHIPK3 (homeodomain interacting protein kinase 3)/miR-125a-5p of NDUFA5 (NADH-ubiquinone oxidoreductase 1 alpha subcomplex 5) in diabetic retinopathy (DR) by integrating public single-cell and bulk RNA-seq data with experimental validation, thereby providing a basis for investigating DR pathogenesis and identifying therapeutic targets.

Methods: scRNA-seq data of DR were obtained from the GEO database, and differential expression of the key gene NDUFA5 was validated using bulk data. Human retinal microvascular endothelial cells (hRMECs) were exposed to 30 mmol/L glucose to establish a high-glucose (HG) cell model. NDUFA5 was knocked down in hRMECs. Cell viability was detected by Cell Counting Kit-8. The levels of oxidative stress indicators (malondialdehyde [MDA], superoxide dismutase [SOD], glutathione peroxidase [GSH-Px]), and inflammatory factors (tumor necrosis factor α [TNF-α], interleukin-6 [IL-6]) were measured using ELISA. Cell migration ability was detected by Transwell assay, and tube formation ability was evaluated by tube formation assay. The expression levels of circHIPK3, miR-125a-5p, and NDUFA5 in cells were detected by RT-qPCR, and the protein expression of NDUFA5 was detected by western blot. The stability of circHIPK3 was assessed by actinomycin D treatment. RNA pull-down and dual-luciferase reporter gene assays were used to verify the binding relationships between circHIPK3/NDUFA5 and miR-125a-5p. The targeting relationship between miR-125a-5p and NDUFA5 was examined by overexpressing miR-125a-5p in HG cells. Combined inhibition of circHIPK3 and miR-125a-5p in HG cells were conducted to validate the above regulatory mechanism. Male C57BL/6J mice (6-8 weeks) were intraperitoneally injected with STZ to establish a DR mouse model. NDUFA5 was inhibited using intravitreal AAV (adeno-associated virus) injection. Retinal tissues were collected for molecular detection.

Results: scRNA-seq analysis revealed that NDUFA5 was specifically enriched in retinal rod cells of DR mice, forming an NDUFA5-enriched rod cell cluster (NDUFA5+RC) enriched in oxidative phosphorylation and HIF-1 signaling pathways. The VEGFA+MUC subpopulation in Müller cells was upregulated in the DR group, enriched in DR-related core pathologic pathways such as VEGF and PI3K/Akt signaling pathways, and bioinformatic prediction enhanced communication by the PTN-SDC4 ligand-receptor pair between VEGFA+MUC and NDUFA5+RC. Bulk RNA-seq validation confirmed that NDUFA5 expression was significantly upregulated in the DR group. Under HG conditions, NDUFA5 and circHIPK3 expression increased in hRMECs, while miR-125a-5p expression decreased. Knockdown of NDUFA5 or overexpression of miR-125a-5p significantly alleviated HG-induced cellular oxidative stress, inflammatory response, and abnormalities in migration and tube formation capacity. Mechanistic validation showed that circHIPK3 is stably expressed in the cytoplasm and is capable of acting as a competitive endogenous RNA (ceRNA) by sponging miR-125a-5p, which is associated with relieved targeted inhibition of NDUFA5. Animal experiments confirmed that NDUFA5 and circHIPK3 were upregulated, while miR-125a-5p was downregulated in the retinae of DR mice. NDUFA5 inhibition significantly reduced retinal vascular leakage and acellular capillary formation, and decreased oxidative stress and inflammation.

Conclusion: In DR, circHIPK3 may sponge miR-125a-5p, relieving NDUFA5 inhibition and contributing to DR progression. NDUFA5 represents a potential therapeutic target for DR.