Identification of metabolism-associated biomarkers with a focus on ketone body pathways in diabetic retinopathy by integrating transcriptomics and single-cell analysis

Background To identify potential biomarkers of ketone body metabolism (KBM) associated with diabetic retinopathy (DR), and to explore possible underlying mechanism between KBM and DR. Methods Differentially expressed genes (DEGs) between DR and healthy normal control (NC) samples were acquired in GSE221521.Then, DEGs and KBM-related genes (KBM-RGs) were interleaved to discern candidate genes, and the key feature genes were derived through two algorithms of machine learning. Next, biomarkers were generated through receiver operating characteristic (ROC) curves analysis and expression validation in GSE221521 and GSE189005.Then, enrichment analysis, immune infiltration and single-cell RNA sequencing analysis were conducted.Finally, experimental validation of biomarkers was conducted through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Results ACACA and WDTC1 were recognized as DR-associated candidate biomarkers. ACACA was enriched in Reactome RNA polymerase II transcription pathway, and WDTC1 was enriched in Reactome eukaryotic translation initiation pathway. Two types of immune cells, namely activated NK cells and monocytes, were significantly different between DR and NC samples in bulk immune infiltration analysis. Next, NK cells and T cells were identified as key cell types based on cell proportion analysis. Both ACACA and WDTC1 were highly expressed in NK cells and T cells. Cell-cell communication patterns changed in the DR group. ACACA and WDTC1 showed stable expression during the differentiation of T cells and NK cells. Finally, RT-qPCR results showed that ACACA and WDTC1 were up-regulated in DR samples. This concurred with the results yielded by bioinformatics analysis. Conclusion This study identified two metabolism-associated biomarkers (ACACA and WDTC1) that may serve as DR-associated candidate biomarkers. These preliminary findings suggest potential molecular leads for further investigation, though prospective validation in larger cohorts is needed.