Identification and Validation of Core Genes for Type 2 Diabetes Mellitus by Integrated Analysis of Single-cell and Bulk RNA-sequencing

Background: The exact mechanisms of type 2 diabetes mellitus (T2DM) remain largely unknown. We intended to authenticate critical genes linked to T2DM progression by tandem single-cell sequencing and general transcriptome sequencing data.

Methods: T2DM single-cell RNA-sequencing data were submitted by the Gene Expression Omnibus (GEO) database and ArrayExpress (EBI), from which gene expression matrices were retrieved. The common cell clusters and representative marker genes were ascertained by principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), CellMarker, and FindMarkers in two datasets (GSE86469 and GSE81608). T2DM-related differentially expressed marker genes were defined by intersection analysis of marker genes and GSE86468-differentially expressed genes. Receiver operating characteristic (ROC) curves were utilized to assign representative marker genes with diagnostic values by GSE86468, GSE29226 and external validation GSE29221, and their prospective target compounds were forecasted by PubChem. Besides, the R package clusterProfiler-based functional annotation was designed to unveil the intrinsic mechanisms of the target genes. At last, western blot was used to validate the alternation of CDKN1C and DLK1 expression in primary pancreatic islet cells cultured with or without 30mM glucose.

Results: Three common cell clusters were authenticated in two independent T2DM single-cell sequencing data, covering neurons, epithelial cells, and smooth muscle cells. Functional ensemble analysis disclosed an intimate association of these cell clusters with peptide/insulin secretion and pancreatic development. Pseudo-temporal trajectory analysis indicated that almost all epithelial and smooth muscle cells were of neuron origin. We characterized CDKN1C and DLK1, which were notably upregulated in T2DM samples, with satisfactory availability in recognizing three representative marker genes in non-diabetic and T2DM samples, and they were also robustly interlinked with the clinical characteristics of patients. Western blot also demonstrated that, compared with control group, the expression of CDKN1C and DLK1 were increased in primary pancreatic islet cells cultured with 30 mM glucose for 48 h. Additionally, PubChem projected 11 and 21 potential compounds for CDKN1C and DLK1, respectively.

Conclusion: It is desirable that the emergence of the 2 critical genes indicated (CDKN1C and DLK1) could be catalysts for the investigation of the mechanisms of T2DM progression and the exploitation of innovative therapies.