High expression of CNOT6L contributes to the negative development of type 2 diabetes

Abstract

Objective: Type 2 diabetes (T2D) is a chronic metabolic disorder characterized by reduced responsiveness of body cells to insulin, leading to elevated blood sugar levels. CNOT6L is involved in glucose metabolism, insulin secretion regulation, pancreatic beta-cell proliferation, and apoptosis. These functions may be closely related to the pathogenesis of T2D. However, the exact molecular mechanisms linking CNOT6L to T2D remain unclear. Therefore, this study aims to elucidate the role of CNOT6L in T2D.

Methods: The T2D datasets GSE163980 and GSE26168 profiles were downloaded from the Gene Expression Omnibusdatabase generated by GPL20115 and GPL6883.The R package limma was used to screen differentially expressed genes (DEGs). A weighted gene co-expression network analysis was performed. Construction and analysis of the protein-protein interaction (PPI) network, functional enrichment analysis, gene set enrichment analysis, and comparative toxicogenomics database (CTD) analysis were performed. Target Scan was used to screen miRNAs that regulate central DEGs. The results were verified by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), and blood glucose measurements in mice.

Results: A total of 1951 DEGs were identified. GO and KEGG enrichment analysis revealed that differentially expressed genes were mainly enriched in the insulin signaling pathway, ECM-receptor interaction, and PPAR signaling pathway. Metascape analysis indicated enrichment primarily in the cAMP signaling pathway and enzyme-linked receptor protein signaling pathway. WGCNA analysis yielded 50 intersecting genes. PPI network construction and algorithm identification identified two core genes (CNOT6L and GRIN2B), among which CNOT6L gene was associated with multiple miRNAs. CTD analysis revealed associations of core genes with type 2 diabetes, diabetic complications, dyslipidemia, hyperglycemia, and inflammation. WB and RT-qPCR results showed that in different pathways, CNOT6L protein and mRNA levels were upregulated in type 2 diabetes.

Conclusion: CNOT6L is highly expressed in type 2 diabetes mellitus, and can cause diabetes complications, inflammation and other physiological processes by regulating miRNA, PPAR and other related signaling pathways, with poor prognosis. CNOT6L can be used as a potential therapeutic target for type 2 diabetes.

Keywords: Bioinformatics; CNOT6L; Molecular targets; Type 2 diabetes.

Fig. 1

Study flow chart.

Fig. 2

Differential gene analysis. A total of 1951 DEGs.

Fig. 3

(A-D) Results of GOKEGG enrichment analysis of DEGs. (A) Biological process analysis. (B) Cellular component analysis. (C) Molecular function analysis. (D) Results of KEGG enrichment analysis. (E-H) Results of GSEA enrichment analysis of DEGs. (E) Biological process analysis. (F) Cellular component analysis. (G) Molecular function analysis. (H) KEGG enrichment analysis.

Fig. 4

Metascape enrichment analysis. (A) Bar graph of enriched terms across input gene lists, colored by p-values. (B) Network of enriched terms: colored by cluster ID, where nodes that share the same cluster ID are typically close to each other. (C) colored by p-value, where terms containing more genes tend to have a more significant p-value.

Fig. 5

WGCNA analysis. (A) β = 3,0.87. β = 3,324.07. (B, C) The hierarchical clustering tree of all genes was constructed, and 17 important modules were generated.

Fig. 6

(A) The heat map of correlation between modules and phenotypes. (B) The scatter map of correlation between GS and MM of related hub genes. (C) The DEGs screened by WGCNA and DEGs was used to obtain venn map. 50 intersection genes were obtained.

Fig. 7

Construction and analysis of protein-protein interaction (PPI) networks. (A) Construct the PPI network of DEGs using STRING online database and utilize Cytoscape software for analysis. (B) Core genes (CNOT6L, GRIN2B) were obtained by merging using Venn diagrams. (C) MCC was used to identify the central gene. (D) MNC was used to identify the central gene. (E) Radiality was used to identify the central gene. (F) EcCentricity was used to identify the central gene.

Fig. 8

CTD analysis. Core genes (CNOT6L, GRIN2B) are associated with type 2 diabetes, diabetes complications, dyslipidemia, hyperglycemia and inflammation.

Fig. 9

Expression of CNOT6L, PPARγ, RXR, PEPCK, AQP7, and GYK in the blood of type 2 diabetes mellitus mice. Protein expression levels were determined by western blotting. A representative blot comparing control (CON), type 2 diabetes (Diabetes), type 2 diabetes CNOT6L gene overexpression (Diabetes-CNOT6L/OE), and type 2 diabetes CNOT6L gene knockout (Diabetes-CNOT6L/KO) groups is shown, with each sample run in duplicate. GAPDH was used as the internal control. The results are presented as mean ± standard deviation of independent 10 experiments.** P < 0.01; *** P < 0.001.

Fig. 10

Expression of ubiquitination-related genes UBC, ILK, and PDK1 in the blood of type 2 diabetes mellitus mice. Protein expression levels were determined by western blotting. A representative blot comparing control (CON), type 2 diabetes (Diabetes), type 2 diabetes CNOT6L gene overexpression (Diabetes-CNOT6L/OE), and type 2 diabetes CNOT6L gene knockout (Diabetes-CNOT6L/KO) groups is shown, with each sample run in duplicate. GAPDH was used as the internal control. The results are presented as mean ± standard deviation of independent 10 experiments. ** P < 0.01.

Fig. 11

Expression of clotting-related genes PAI-1, vWF, SFMC, and TAFI in the blood of type 2 diabetes mellitus mice. Protein expression levels were determined by western blotting. A representative blot comparing control (CON), type 2 diabetes (Diabetes), type 2 diabetes CNOT6L gene overexpression (Diabetes-CNOT6L/OE), and type 2 diabetes CNOT6L gene knockout (Diabetes-CNOT6L/KO) groups is shown, with each sample run in duplicate. GAPDH was used as the internal control. The results are presented as mean ± standard deviation of independent 10 experiments. ** P < 0.01.

Fig. 12

Expression of clotting-related genes ACBP, CYP7A1, CYP27, FABP1, OLR1, CPT1, CPT2, and LCAD in the blood of type 2 diabetes mellitus mice. Protein expression levels were determined by western blotting. A representative blot comparing control (CON), type 2 diabetes (Diabetes), type 2 diabetes CNOT6L gene overexpression (Diabetes-CNOT6L/OE), and type 2 diabetes CNOT6L gene knockout (Diabetes-CNOT6L/KO) groups is shown, with each sample run in duplicate. GAPDH was used as the internal control. The results are presented as mean ± standard deviation of independent 10 experiments. ** P < 0.01.

Fig. 13

Expression of VEGF and ANG in the blood of type 2 diabetes mellitus mice. Protein expression levels were determined by western blotting. A representative blot comparing control (CON), type 2 diabetes (Diabetes), type 2 diabetes CNOT6L gene overexpression (Diabetes-CNOT6L/OE), and type 2 diabetes CNOT6L gene knockout (Diabetes-CNOT6L/KO) groups is shown, with each sample run in duplicate. GAPDH was used as the internal control. The results are presented as mean ± standard deviation of independent 10 experiments. ** P < 0.01.

Fig. 14

Expression of ICAM-1, VCAM-1, P-selectin, E-selectin, MCP-1, MMP-2, and MMP-9 in the blood of type 2 diabetes mellitus mice. Protein expression levels were determined by western blotting. A representative blot comparing control (CON), type 2 diabetes (Diabetes), type 2 diabetes CNOT6L gene overexpression (Diabetes-CNOT6L/OE), and type 2 diabetes CNOT6L gene knockout (Diabetes-CNOT6L/KO) groups is shown, with each sample run in duplicate. GAPDH was used as the internal control. The results are presented as mean ± standard deviation of independent 10 experiments. ** P < 0.01.

Fig. 15

Relative mRNA expression of CNOT6L in the blood of type 2 diabetes mellitus mice. Expression levels were determined by RT-qPCR for the control (CON), type 2 diabetes (Diabetes), type 2 diabetes CNOT6L gene overexpression (Diabetes-CNOT6L/OE), and type 2 diabetes CNOT6L gene knockout (Diabetes-CNOT6L/KO) groups. The results are presented as mean ± standard deviation of independent 10 experiments. ** P < 0.01.

Fig. 16

Blood glucose levels in type 2 diabetes mellitus mice. Glucose levels were determined for the control, type 2 diabetes (Diabetes), type 2 diabetes CNOT6L gene overexpression (Diabetes-CNOT6L/OE), and type 2 diabetes CNOT6L gene knockout (Diabetes-CNOT6L/KO) groups. The results are presented as mean ± standard deviation of independent 10 experiments. ** P < 0.01.