MicroRNA-409-3p/BTG2 signaling axis improves impaired angiogenesis and wound healing in obese mice

Abstract

Wound healing is facilitated by neoangiogenesis, a complex process that is essential to tissue repair in response to injury. MicroRNAs are small, noncoding RNAs that can regulate the wound healing process including stimulation of impaired angiogenesis that is associated with type-2 diabetes (T2D). Expression of miR-409-3p was significantly increased in the nonhealing skin wounds of patients with T2D compared to the non-wounded normal skin, and in the skin of a murine model with T2D. In response to high glucose, neutralization of miR-409-3p markedly improved EC growth and migration in human umbilical vein endothelial cells (HUVECs), promoted wound closure and angiogenesis as measured by increased CD31 in human skin organoids, while overexpression attenuated EC angiogenic responses. Bulk mRNA-Seq transcriptomic profiling revealed BTG2 as a target of miR-409-3p, where overexpression of miR-409-3p significantly decreased BTG2 mRNA and protein expression. A 3' untranslated region (3'-UTR) luciferase assay of BTG2 revealed decreased luciferase activity with overexpression of miR-409-3p, while inhibition had opposite effects. Mechanistically, in response to high glucose, miR-409-3p deficiency in ECs resulted in increased mTOR phosphorylation, meanwhile BTG-anti-proliferation factor 2 (BTG2) silencing significantly decreased mTOR phosphorylation. Endothelial-specific and tamoxifen-inducible miR-409-3p knockout mice (MiR-409^IndECKO ) with hyperglycemia that underwent dorsal skin wounding showed significant improvement of wound closure, increased blood flow, granulation tissue thickness (GTT), and CD31 that correlated with increased BTG2 expression. Taken together, our results show that miR-409-3p is a critical mediator of impaired angiogenesis in diabetic skin wound healing.

Keywords: BTG2; angiogenesis; endothelial cells; microRNAs; obesity; wound healing.

FIGURE 1. MiR-409–3p expression in human and murine skin samples with T2D.

(A, B, C) Expression of miR-409–3p in (A) the non-healing wounds and normal skin of patients with T2D, (B) dorsal skin wound samples of WT or db/db mice, and (C) across various organs of WT mice. (D) MiR-409–3p expression in HUVECs treated with 30 mM mannitol or glucose for indicated durations. Data representative of n = 7–8 patients per condition (A), n = 4–6 per condition (B-D). Statistical significance was determined by unpaired student’s t-test or two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate ± SEM.

FIGURE 2. MiR-409–3p regulates EC angiogenic function and wound healing in response to glucose.

HUVECs transfected with non-specific control (NS_m) or miR-409–3p mimic (miR-409–3p_m) or non-specific inhibitor control (NS_i) or miR-409–3p inhibitor (miR-409–3p_i) and treated with 30 mM mannitol or glucose were subjected to (A) scratch migration and (B) growth assays. Punch biopsies of human skin transfected with NS_m or miR-409–3p_m, or NS_i or miR-409–3p_i were evaluated for (C) wound closure at the indicated time points and (D) angiogenesis as measured by CD31 staining across the wound edges, scale bars = 50 μm. Statistical significance was determined by unpaired student’s t-test and based on comparison with respective control group. *p<0.05, **p<0.01, ***p<0.001. n= 6–8 replicates per condition. Error bars indicate ± SEM.

FIGURE 3. BTG2 is a target of miR-409–3p.

(A-C) Genome-wide RNA-Seq analyses were performed from HUVECs transfected with NS_m or miR-409–3p_m. (A) Gene Ontology analysis depicting top cellular and physiologic functions. (B) Volcano plot of differentially expressed genes with FDR corrected p-value <0.01 and Fold Change (FC) >1.4. (C) Heatmap of top 10 most downregulated genes. (D-E) HUVECs or human skin organoids transfected with NS_m or miR-409–3p_m, NS_i or miR-409–3pi were subjected to (D) RT-qPCR to measure BTG2 mRNA or (E) Western blot analysis to measure BTG2 protein expression. (F) Luciferase activity of BTG2 3’-untranslated region (UTR) normalized to protein concentration in HEK293T cells. Statistical significance was determined by unpaired student’s t-test or two-way ANOVA, based on comparison with the respective non-specific control group. *p<0.05, **p<0.01, ***p<0.001. n= 6–8 replicates per condition. Error bars indicate ± SEM.

FIGURE 4. MiR-409–3p knockdown regulates mTOR signaling pathway in response to glucose.

(A) Gene network visualization of the genome-wide RNA-Seq analyses from ECs overexpressing miR-409–3p or the non-specific control predicted mTOR as a candidate downstream signaling component. (B, C) Western blot analyses of phosphorylated-mTOR (p-mTOR), mTOR, β-actin, and GAPDH in (B) HUVECs or (C) human skin organoids transfected with NS_i or miR-409–3p_i and treated with 30 mM glucose. Statistical significance was determined by unpaired student’s t-test or two-way ANOVA, based on a comparison with respective non-specific control group. *p<0.05, **p<0.01. ns indicates not significant. n= 4–6 replicates per condition. Error bars indicate ± SEM.

FIGURE 5. BTG2 neutralization mimics miR-409–3p’s functional effects in vitro.

HUVECs transfected with negative control siRNA (Ctrl_siRNA) or BTG2 siRNA (BTG2_siRNA) and treated with 30 mM glucose were subjected to (A) EC migration, (B) EC growth, and (C) Western blot analysis for p-mTOR, mTOR, and β-actin protein expression. Statistical significance was determined by unpaired student’s t-test or two-way ANOVA, based on comparison with indicated control group. *p < 0.05, **p < 0.01, ***p < 0.001. ns indicates not significant. n= 7–9 replicates per condition. Error bars indicate ± SEM.

FIGURE 6. Endothelial specific miR-409–3p knockout promotes wound healing and angiogenesis in vivo.

Dorsal skin injury was performed on MiR409^Ind-EKCO and MiR409^fl/fl control mice after 8 weeks of high fat, high sucrose diet. (A) Schema of experimental procedure. Wound analyses included LSCI blood flow measurements (B), % Wound closure (C), scale bars = 2mm. (D) Granulation tissue thickness (GTT) as quantified in skin wound samples H&E staining, scale bars= 300 μm. (E) Angiogenesis quantified in wound sections stained for CD31, scale bars = 100 μm, inset = 12.5 μm. (F) Relative mRNA expression levels of BTG2 in skin ECs 7 days post-wounding. (G) BTG2 levels are quantified in mouse skin wounds via immunofluorescence staining and confocal microscopy scale bars = 100 μm, inset = 12.5 μm. Statistical significance was determined by unpaired student’s t-test or two-way ANOVA, based on comparison with indicated control group. *p < 0.05, **p < 0.01, ***p < 0.001. ns indicates not significant. n=6–8 mice per condition unless noted otherwise. Error bars indicate ± SEM.

FIGURE 7. The mechanism of miR-409–3p’s role in wound healing and angiogenesis.

In patients with T2D and non-healing skin wounds, miR-409–3p levels were significantly elevated within the wound tissue while BTG2 expression was decreased compared to the non-wounded skin samples. When miR-409–3p expression was neutralized in human skin organoids using miR-409–3p inhibitors or genetically knocked-down in ECs of a tamoxifen inducible transgenic mouse model; reduction in miR-409–3p expression resulted in increased BTG2 expression, mTOR phosphorylation, angiogenesis and skin wound healing.