The MSC-Derived Exosomal lncRNA H19 Promotes Wound Healing in Diabetic Foot Ulcers by Upregulating PTEN via MicroRNA-152-3p

Abstract

Mesenchymal stem cells (MSCs) have been reported to hold promise to accelerate the wound-healing process in diabetic foot ulcer (DFU) due to the multilineage differentiation potential. Hence, this study intended to explore the wound healing role of MSC-derived exosomes containing long noncoding RNA (lncRNA) H19 in DFU. lncRNA H19 was predicated to bind to microRNA-152-3p (miR-152-3p), which targeted phosphatase and tensin homolog (PTEN) deleted on chromosome ten. Fibroblasts in DFU samples exhibited highly expressed miR-152-3p and poorly expressed lncRNA H19 and PTEN, along with an activated phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (Akt1) signaling pathway. The fibroblasts were cocultured with lncRNA H19-transfected MSCs and MSC-derived exosomes to assess the effect of the lncRNA H19/miR-152-3p/PTEN axis on the biological activities and inflammation in fibroblasts. Mouse models of DFU were developed by streptozotocin, which was injected with MSC-derived exosomes overexpressing lncRNA H19. lncRNA H19 in MSCs was transferred through exosomes to fibroblasts, the mechanism of which improved wound healing in DFU, corresponded to promoted fibroblast proliferation and migration, as well as suppressed apoptosis and inflammation. Wound healing in mice with DFU was facilitated following the injection of MSC-derived exosomes overexpressing lncRNA H19. Taken together, MSC-derived exosomal lncRNA H19 prevented the apoptosis and inflammation of fibroblasts by impairing miR-152-3p-mediated PTEN inhibition, leading to the stimulated wound-healing process in DFU.

Keywords: PTEN; diabetic foot ulcer; exosome; long noncoding RNA H19; mesenchymal stem cells; microRNA-152-3p.

Figure 1

Culture and Characterization of Fibroblasts (A) Morphological observation of fibroblasts (200×). (B) Immunohistochemical analysis of fibroblasts (200×).

Figure 2

miR-152-3p Exerts Effects on Proliferation, Migration, and Apoptosis of Fibroblasts by Targeting PTEN (A) Expression of miR-152-3p in normal foot tissue (n = 40) and foot tissue from patients with DFU (n = 69), determined by qRT-PCR. (B) Predicted binding sites between miR-152-3p and PTEN by the starBase database. (C) mRNA expression of PTEN in normal foot tissue (n = 40) and foot tissue from patients with DFU (n = 69), determined by qRT-PCR. (D) Correlation between miR-152-3p and PTEN analyzed by Pearson correlation coefficient. (E) Luciferase activity of PTEN-WT and PTEN-Mut detected by dual-luciferase reporter gene assay. (F) mRNA expression of PTEN in fibroblasts after transfection, determined by qRT-PCR. (G) Protein bond diagram of PTEN in fibroblasts after transfection determined by western blot analysis. (H) Relative protein expression of PTEN normalized to GAPDH in fibroblasts after transfection determined by western blot analysis. (I) Fibroblast proliferation detected by EdU assay (200×). (J) Fibroblast migration detected by Transwell assay (200×). (K) Fibroblast apoptosis detected by TUNEL assay (200×). **p < 0.01 versus the normal group (normal foot tissue) or the mimic-NC group (fibroblasts treated with mimic-NC); ##p < 0.01 versus the inhibitor-NC group (fibroblasts treated with inhibitor-NC); &&p < 0.01 versus the oe-NC group (fibroblasts treated with oe-NC). Measurement data were expressed as mean ± SD. Comparison between two groups was conducted using independent sample t test. One-way ANOVA was used for data comparison among multiple groups, followed by Tukey’s post hoc test. The experiment was repeated independently three times.

Figure 3

Overexpressed lncRNA H19 Competitively Binding to miR-152-3p Affects Fibroblast Proliferation, Migration, and Apoptosis (A) Binding sites between lncRNA H19 and miR-152-3p. (B) Subcellular localization of lncRNA H19 detected by FISH (400×). (C) Luciferase activity of H19-WT and H19-Mut detected by dual-luciferase reporter gene assay. (D) Relative enrichment of lncRNA H19 detected by RNA pull-down. (E) Relative enrichment of Ago2 by lncRNA H19 and miR-152-3p detected by RIP assay. (F) lncRNA H19 and miR-152-3p expression and mRNA expression of PTEN, determined by qRT-PCR. (G) Fibroblast proliferation detected by EdU assay (200×). (H) Fibroblast migration detected by Transwell assay (200×). (I) Fibroblast apoptosis detected by TUNEL assay (200×). **p < 0.01 versus the oe-NC group (fibroblasts treated with oe-NC); ##p < 0.01 versus the sh-NC group (fibroblasts treated with sh-NC). Measurement data were expressed as mean ± SD. Comparison between two groups was conducted using independent sample t test. One-way ANOVA was used for data comparison among multiple groups, followed by Tukey’s post hoc test. The experiment was repeated independently three times.

Figure 4

Characterization of MSCs and MSC-exo (A) Morphological observation of MSCs (200×). (B) Surface marker molecules of MSCs detected by flow cytometry. (C) Representative images of osteocyte, adipocyte, and chondrocyte differentiation of MSCs analyzed using cytochemical staining with alizarin red (a), oil red O (b), and Alcian blue (c) (400×). (D) Morphology change of MSC-exo detected by TEM (scale bar, 200 nm). (E) The size distribution of the MSC-exo examined using dynamic light scattering. (F) Surface marker (CD63) of exosomes detected by flow cytometry. (G) The positive markers for exosomes, including CD63, CD81, TSG101, and HSP70, were detected in MSC-exo using western blot analysis, whereas the negative marker GRP94 was not detected. The experiment was repeated independently three times.

Figure 5

MSCs Regulate the Expression of miR-152-3p and PTEN in Fibroblasts through MSC-exo Carrying lncRNA H19 (A) The uptake of MSC-exo by fibroblasts observed under an inverted microscope (400×). (B) Expression of lncRNA H19 in MSCs and exosomes, determined by qRT-PCR. (C) Secretion of exosomes in medium with exosome inhibitor added, observed under TEM (scale bar, 200 nm). (D) Expression of lncRNA H19, miR-152-3p, and PTEN, determined by qRT-PCR. (E) Protein bond diagram of PTEN determined by western blot analysis. (F) Relative protein expression of PTEN normalized to GAPDH, determined by western blot analysis. **p < 0.01 versus the control group (fibroblasts without any treatment); ##p < 0.01 versus the MSC oe-NC group (MSCs treated with oe-NC); &&p < 0.01 versus the MSC sh-NC group (MSCs treated with sh-NC). Measurement data were expressed as mean ± SD. One-way ANOVA was used for data comparison among multiple groups, followed by Tukey’s post hoc test. The experiment was repeated independently three times.

Figure 6

MSC-exo Carrying lncRNA H19 Regulates Fibroblast Proliferation, Migration, and Apoptosis via the PI3K/AKT Signaling Pathway (A) Fibroblast proliferation detected by EdU assay (200×). (B) Fibroblast migration detected by Transwell assay (200×). (C) Fibroblast apoptosis detected by TUNEL assay (200×). (D) Protein bond diagram of PTEN, p85 PI3K, and AKT, as well as the extent of AKT phosphorylation determined by western blot analysis. (E) Relative protein expression of PTEN, p85 PI3K, and AKT, as well as the extent of AKT phosphorylation normalized to GAPDH, determined by western blot analysis. **p < 0.01 versus the control group (fibroblasts without any treatment); ##p < 0.01 versus the MSC-exo-oe-NC group (MSC-exo treated with oe-NC); &&p < 0.01 versus the MSC-exo-sh-NC (MSC-exo treated with sh-NC). Measurement data were expressed as mean ± SD. Comparison among multiple groups was conducted using one-way ANOVA, followed by Tukey’s post hoc test. The experiment was repeated independently three times.

Figure 7

MSC-exo Carrying lncRNA H19 Promotes Wound Healing in Mice with DFU (A) Representative images displaying the wound-healing process of mice with DFU. (B) Wound area of mice with DFU. (C) Edge tissues of wound identified by H&E staining (200×). (D) miR-152-3p expression and mRNA expression of PTEN in wound tissues, determined by qRT-PCR. (E) Protein bond diagram of PTEN, p85 PI3K, and AKT, as well as the extent of AKT phosphorylation determined by western blot analysis. (F) Relative protein expression of PTEN, p85 PI3K, and AKT, as well as the extent of AKT phosphorylation normalized to GAPDH, determined by western blot analysis. (G) Levels of inflammatory factors (IL-1β, TNF-α, and IL-10), determined by ELISA. (H and I) Relative protein expression of angiogenesis-related factors (VEGF, TNF-β1, α-SMA, and collagen I) normalized to GAPDH, determined by western blot analysis. (J) Cell apoptosis detected by TUNEL assay (200×). **p < 0.01 versus the control group (n = 12, DFU mice without any treatment). Measurement data were expressed as mean ± SD. Comparison among multiple groups was conducted using one-way ANOVA, followed by Tukey’s post hoc test. Repeated-measures ANOVA was applied for data comparison among multiple groups at different time points, followed by Tukey’s post hoc test (B).