Gene expression profiling of human umbilical vein endothelial cells overexpressing CELF2 as diagnostic targets in diabetes-induced erectile dysfunction

Abstract

Background: Erectile dysfunction (ED) is a common complication of diabetes mellitus (DM), and because of its complex neurovascular etiology, the associated molecular pathogenic mechanisms are not fully understood. This study investigated the important functions and potential molecular regulatory roles of CELF2 in DMED.

Methods: An in vitro HUVEC model with CELF2 overexpression was successfully established via transfection with a CELF2-overexpressing lentiviral vector. The effects of CELF2 overexpression on cell proliferation and angiogenesis were assessed via CCK-8 and angiogenesis assays. RNA sequencing was employed to evaluate the gene expression profiles and alternative splicing events regulated by CELF2. An RNA-sequencing assay was performed to evaluate gene expression profiles and alternative splicing genes in HUVECs overexpressing CELF2, and an integration analysis was combined with GSE146078 data to detect potential target genes related to DMED.

Results: The expression of genes related to angiogenesis and the immune response significantly increased with CELF2 overexpression, and the four hub genes associated with alternative splicing in aging and angiogenesis were CXCL2, CXCL10, IL-1A and IL-6.

Conclusion: CELF2 appears to be a key factor in DMED, influencing gene expression and alternative splicing related to angiogenesis and immune responses. The identified hub genes (CXCL2, CXCL10, IL-1A, and IL-6) are closely related to DMED and warrant further investigation to understand the underlying mechanisms and potential therapeutic implications.

Keywords: CELF2; RBPs; RNA sequencing; diabetes mellitus; erectile dysfunction.

FIGURE 1

CELF2 overexpression accelerates the proliferation and angiogenesis of HUVECs. (A) The histogram shows the RT‒qPCR results of CELF2-overexpressing HUVECs. The error bars represent the means ± SEMs. ****P value <0.0001. (B) Western blot analysis revealed that CELF2 was successfully overexpressed in HUVECs. (C) The histogram shows the proliferation of CELF2-overexpressing cells after 72 h. (D) The diagram shows the angiogenic experimental results of CELF2 overexpression after 20 h. The left panel shows the results of angiogenesis in cells, and the right panel shows the quantitative results of CELF2 overexpression after 20 h.

FIGURE 2

CELF2 regulates gene expression in HUVECs. (A) PCA based on the FPKM values of all detected genes. The ellipse for each group is the confidence ellipse. (B) Volcano plot showing all differentially expressed genes (DEGs) between the OE-CELF2 and NC samples. (C) Hierarchical clustering heatmap showing the expression levels of all DEGs. (D) Histogram showing the most enriched GO biological process results for the upregulated genes and downregulated genes. (E) Bar plot showing the expression patterns and significant differences in DEGs among the four upregulated genes and four downregulated genes. The error bars represent the means ± SEMs. ***P value <0.001. **P value <0.01. *P value <0.05.

FIGURE 3

CELF2 influences alternative splicing of genes in HUVECs. (A) Bar plot showing the percentages of each type of upregulated or downregulated CELF2-regulated alternative splicing event (RASE). (B) Boxplot showing the standard deviation of the ratio of each type of CELF2-regulated alternative splicing event (RASE) in all samples. (C) Scatter plot showing the most enriched GO biological process results of the regulated alternative splicing genes (RASGs). (D) Schematic diagrams depicting the regulation of alternative splicing genes by CELF2. The error bars represent the means ± SEMs. ***P value <0.001. **P value <0.01. *P value <0.05.

FIGURE 4

DEG Data of GSE146078. (A) PCA based on the FPKM values of all detected genes. The ellipse for each group is the confidence ellipse. (B) Bar plot showing the expression pattern and significant difference in Celf2 expression. The error bars represent the means ± SEMs. ** P value < 0.01. (C) Scatter plot showing the most enriched GO biological process results for the upregulated genes. (D) Scatter plot showing the most enriched GO biological process results for the downregulated genes. The error bars represent the means ± SEMs. ** P value < 0.01.

FIGURE 5

Verification of key target gene expression of CELF2. (A) Venn diagram showing the overlapping DEGs between HUVECs and GSE146078. (B) Scatter plot showing the most enriched GO biological process results of the overlapping DEGs. (C) The network diagram shows the hub genes selected from the overlapping DEGs through the PPI (confidence level 0.4) and MCODE interaction analyses. (D) Bar plot showing the expression patterns and significant differences of the overlapping DEGs. The error bars represent the means ± SEMs. ***P value <0.001. **P value <0.01. *P value <0.05.