MicroRNA-133b Inhibition Restores EGFR Expression and Accelerates Diabetes-Impaired Wound Healing

Abstract

Diabetic foot ulcers (DFUs) are caused by impairments in peripheral blood vessel angiogenesis and represent a great clinical challenge. Although various innovative techniques and drugs have been developed for treating DFUs, therapeutic outcomes remain unsatisfactory. Using the GEO database, we obtained transcriptomic microarray data for DFUs and control wounds and detected a significant downregulation of epidermal growth factor receptor (EGFR) in DFUs. We cultured human umbilical vein endothelial cells (HUVECs) and noted downregulated EGFR expression following high-glucose exposure in vitro. Further, we observed decreased HUVEC proliferation and migration and increased apoptosis after shRNA-mediated EGFR silencing in these cells. In mice, EGFR inhibition via focal EGFR-shRNA injection delayed wound healing. Target prediction analysis followed by dual-luciferase reporter assays indicated that microRNA-133b (miR-133b) is a putative upstream regulator of EGFR expression. Increased miR-133b expression was observed in both glucose-treated HUVECs and wounds from diabetes patients, but no such change was observed in controls. miR-133b suppression enhanced the proliferation and angiogenic potential of cultured HUVECs and also accelerated wound healing. Although angiogenesis is not the sole mechanism affected in DFU, these findings suggest that the miR-133b-induced downregulation of EGFR may contribute to delayed wound healing in diabetes. Hence, miR-133b inhibition may be a useful strategy for treating diabetic wounds.

Figure 1

EGFR expression is decreased in DFUs. (a) Hierarchical clustering heat map constructed using GSE80178 data. (b) Volcano plot based on GSE80178 data. (c) Degree centrality analysis of the top 50 degree-filtered genes from the DEG PPI network and corresponding chromosomal positions. The top 10 hub genes are highlighted in red. (d) Distribution of degree, betweenness, and closeness of the top 10 hub genes. (e) Enrichment analysis findings for the top 10 genes in the PPI network. (f) Expression of EGFR in skin tissues from non-DFU (n = 6) and DFU patients (n = 6) measured using qRT-PCR analysis.

Figure 2

High-glucose exposure decreases EGFR expression in HUVECs. (a) Relative EGFR mRNA levels in peripheral blood from control volunteers and DFU patients (n = 12 per group). (b) Changes in relative EGFR mRNA levels in HUVECs, 4 and 24 h following exposure to 20 mM D-glucose. (c, d) Relative EGFR mRNA levels in wounds from nondiabetic (c) and diabetic mice (d) at 0, 3, and 7 d postwound induction.

Figure 3

EGFR inhibition impairs HUVEC functions. (a) Relative EGFR mRNA expression in control, shNC, and shEGFR HUVECs. (b) Effect of EGFR shRNA treatment on HUVEC proliferation (CCK-8 assay). (c) qRT-PCR results showing Cyclin D1 and Cyclin D3 expression in HUVECs treated with EGFR shRNA. (d) qRT-PCR results showing Bcl-2 and Bax expression in HUVECs treated with EGFR shRNA. (e, f) Transwell migration assay findings depicting the effect of EGFR shRNA on HUVEC migration. Scale bar: 100 μm.

Figure 4

EGFR inhibition delays wound healing in vivo. (a) Wound closure at various time points after different treatments. (b) Wound closure rates for the three experimental treatments.

Figure 5

miR-133b is a potential regulator of EGFR expression. (a) Identification of miR-133b as a putative regulator of EGFR expression based on miRTarBase, TargetScan, and miRWalk target prediction analyses. (b, c) Luciferase reporter assay demonstrating that miR-133b binds to the 3′UTR of the EGFR mRNA. (d) Relative miR-133b levels in control and agomiR-NC-, agomiR-133b-, antagomiR-NC-, and antagomiR-133b-transfected HUVECs (qRT-PCR). (e) qRT-PCR for assessing the effect of miR-133b on EGFR expression. (f) Changes in relative miR-133b levels following exposure of HUVECs to 20 mM D-glucose for 4 and 24 h. (g, h) Relative miR-133b expression in wounds from nondiabetic (g) and diabetic mice (h) at 0, 3, and 7 d postwound induction.

Figure 6

Suppression of miR-133b enhances HUVEC functions. (a) qRT-PCR for miR-133b expression in HUVECs transfected with miR-133b agonist and antagonist mimics. (b) CCK-8 proliferation assay showing HUVECs with enhanced and suppressed miR-133b expression. (c, d) Analysis of Bcl-2 and Bax expression (c) and Cyclin D1 and Cyclin D3 expression (d) using qRT-PCR. (e) Transwell migration assay illustrating the effect of miR-133b inhibition on cell migration. Scale bar: 100 μm.

Figure 7

Administration of antagomiR-133b accelerates wound healing in vivo. (a) General condition of wounds after treatment with PBS (control), agomiR-133b, and antagomiR-133b. (b) Wound closure rates. (c) Expression of miR-133b in wound samples.