MX1 and UBE2L6 are potential metaflammation gene targets in both diabetes and atherosclerosis

Abstract

Background: The coexistence of diabetes mellitus (DM) and atherosclerosis (AS) is widespread, although the explicit metabolism and metabolism-associated molecular patterns (MAMPs) responsible for the correlation are still unclear.

Methods: Twenty-four genetically wild-type male Ba-Ma mini pigs were randomly divided into five groups distinguished by different combinations of 90 mg/kg streptozotocin (STZ) intravenous injection and high-cholesterol/lipid (HC) or high-lipid (HL) diet feeding for 9 months in total. Pigs in the STZ+HC and STZ+HL groups were injected with STZ first and then fed the HC or HL diet for 9 months. In contrast, pigs in the HC+STZ and HL+STZ groups were fed the HC or HL diet for 9 months and injected with STZ at 3 months. The controls were only fed a regular diet for 9 months. The blood glucose and abdominal aortic plaque observed through oil red O staining were used as evaluation indicators for successful modelling of DM and AS. A microarray gene expression analysis of all subjects was performed.

Results: Atherosclerotic lesions were observed only in the HC+STZ and STZ+HC groups. A total of 103 differentially expressed genes (DEGs) were identified as common between them. The most significantly enriched pathways of 103 common DEGs were influenza A, hepatitis C, and measles. The global and internal protein-protein interaction (PPI) networks of the 103 common DEGs consisted of 648 and 14 nodes, respectively. The top 10 hub proteins, namely, ISG15, IRG6, IRF7, IFIT3, MX1, UBE2L6, DDX58, IFIT2, USP18, and IFI44L, drive aspects of DM and AS. MX1 and UBE2L6 were the intersection of internal and global PPI networks. The expression of MX1 and UBE2L6 was 507.22 ± 342.56 and 96.99 ± 49.92 in the HC+STZ group, respectively, which was significantly higher than others and may be linked to the severity of hyperglycaemia-related atherosclerosis. Further PPI network analysis of calcium/micronutrients, including MX1 and UBE2L6, consisted of 58 and 18 nodes, respectively. The most significantly enriched KEGG pathways were glutathione metabolism, pyrimidine metabolism, purine metabolism, and metabolic pathways.

Conclusions: The global and internal PPI network of the 103 common DEGs consisted of 648 and 14 nodes, respectively. The intersection of the nodes of internal and global PPI networks was MX1 and UBE2L6, suggesting their key role in the comorbidity mechanism of DM and AS. This inference was partly verified by the overexpression of MX1 and UBE2L6 in the HC+STZ group but not others. Further calcium- and micronutrient-related enriched KEGG pathway analysis supported that MX1 and UBE2L6 may affect the inflammatory response through micronutrient metabolic pathways, conceptually named metaflammation. Collectively, MX1 and UBE2L6 may be potential common biomarkers for DM and AS that may reveal metaflammatory aspects of the pathological process, although proper validation is still needed to determine their contribution to the detailed mechanism.

Keywords: Atherosclerosis; Biomarker signatures; Diabetes; Differentially expressed genes; Protein–protein interaction.

Figure 1. The common DEGs between the HC+STZ, STZ+HC, HL+STZ, and STZ+HL groups.

A total of 103 transcripts were identified as common between the DEGs of the HC+STZ and STZ+HC groups. A total of 915 and 412 DEGs were specifically expressed in the HC+STZ and STZ+HC groups, respectively. HC+STZ: pigs were fed a high-cholesterol/lipid diet for 9 months and injected with STZ at 3 months (n = 4). STZ+HC: pigs were injected with STZ first and then fed a high-cholesterol/lipid diet for 9 months (n = 3). HL+STZ: pigs were fed a high-lipid diet for 9 months and injected with STZ at 3 months (n = 4). STZ+HL: pigs were injected with STZ first and then fed a high-lipid diet for 9 months (n = 5). The biological replicate numbers of the control, HC+STZ, STZ+HC, HL+STZ, and STZ+HL groups were 4, 4, 3, 4, and 5, respectively. The technical replicate number of all samples was 2.

Figure 2. Heatmap of the DEGs in the different groups.

Annotations on the top of the heatmap show clustering of the samples. The Z score represents the normalized value of the differential gene FPKM. The closer the color is to rose red, the higher the gene expression is. In contrast, the closer the color is to blue, the lower the gene expression is. HC+STZ: pigs were fed a high-cholesterol/lipid diet for 9 months and injected with STZ at 3 months (n = 4). STZ+HC: pigs were injected with STZ first and then fed a high-cholesterol/lipid diet for 9 months (n = 3). HL+STZ: pigs were fed a high-lipid diet for 9 months and injected with STZ at 3 months (n = 4). STZ+HL: pigs were injected with STZ first and then fed a high-lipid diet for 9 months (n = 5).

Figure 3. The BP, CC, and MF terms or pathways enriched in the 915 genes from the HC+STZ group were analysed by GO and KEGG analyses.

HC+STZ: pigs were fed a high-cholesterol/lipid diet for 9 months and injected with STZ at 3 months (n = 4). GeneRatio, the ratio of the number of genes annotated to this entry in the selected gene set to the total number of genes annotated to this entry in this species. Padj, also known as the p value correction value.

Figure 4. The BP, CC, and MF terms or pathways enriched in the 412 genes from the STZ+HC group were analysed by GO and KEGG analyses.

STZ+HC: pigs were injected with STZ first and then fed a high-cholesterol/lipid diet for 9 months (n = 3). GeneRatio, the ratio of the number of genes annotated to this entry in the selected gene set to the total number of genes annotated to this entry in this species. Padj, also known as the p value correction value.

Figure 5. The BP, CC, and MF terms or pathways enriched in the 103 genes common to the HC+STZ and STZ+HC groups were analysed by GO and KEGG analyses.

HC+STZ: pigs were fed a high-cholesterol/lipid diet for 9 months and injected with STZ at 3 months (n = 4). STZ+HC: pigs were injected with STZ first and then fed a high-cholesterol/lipid diet for 9 months (n = 3). GeneRatio, the ratio of the number of genes annotated to this entry in the selected gene set to the total number of genes annotated to this entry in this species. Padj, also known as the p value correction value.

Figure 6. PPI analysis of 103 common DEGs of HC+STZ vs control and STZ+HC vs control.

(A) The global PPI network, which consists of 648 nodes and 2,106 edges, was constructed by retrieving the global and internal interactions of the 103 common DEGs from the STRING database. (B) The top 10 hub proteins in the global PPI network were screened using the CytoHubba plugin of the Cytoscape software using the Degree method. The darker the colour is in the color gradient, the higher the coreness is. (C) Internal PPI network of 103 common DEGs, consisting of 14 nodes and 31 edges. HC+STZ: pigs were fed a high-cholesterol/lipid diet for 9 months and injected with STZ at 3 months (n = 4). STZ+HC: pigs were injected with STZ first and then fed a high-cholesterol/lipid diet for 9 months (n = 3).

Figure 7. The expression of MX1 and UBE2L6 links DM and AS.

(A and B) The expression of MX1 and UBE2L6 was significantly increased in the HC+STZ group compared with the other groups (*p < 0.05; **p < 0.01). Data shown as scatter plots with mean ± standard deviation. (C and D) The association between the presence or severity of AS of the abdominal aorta and overexpression of MX1 or UBE2L6 related to higher levels of glucose was traced by a Sankey diagram. The numbers in each colour bar represent the number of samples for the corresponding classification. The numbers in parentheses after each group label are their fasting blood glucose values (measured in mmol/l, expressed as the mean). HC+STZ: pigs were fed a high-cholesterol/lipid diet for 9 months and injected with STZ at 3 months (n = 4). STZ+HC: pigs were injected with STZ first and then fed a high-cholesterol/lipid diet for 9 months (n = 3). HL+STZ: pigs were fed a high-lipid diet for 9 months and injected with STZ at 3 months (n = 4). STZ+HL: pigs were injected with STZ first and then fed a high-lipid diet for 9 months (n = 5). At higher MXI/UBE2L6 levels, the expression levels of the corresponding genes were significantly higher than those in the control group. NS. MXI/UBE2L6, the expression levels of the corresponding genes were similar to those in the control group. AS (−), no fatty streak or atherosclerotic plaque; AS (+), fatty streak; AS (++), atherosclerotic plaque is less than half of the lumen; AS (+++), atherosclerotic plaque is more than half of the lumen.

Figure 8. PPI analysis of calcium and micronutrient potential candidate genes (n = 13,802 and 382) with MX1 and UBE2L6.

(A) The calcium-related PPI network in which MX1 and UBE2L6 participate contained 58 nodes and 424 edges. (B) The micronutrient-related PPI network in which MX1 and UBE2L6 participate contained 18 nodes and 58 edges.