Int J Stem Cells.  2022 Nov;15(4):359-371. 10.15283/ijsc21135.

MSCs-Derived miR-150-5p-Expressing Exosomes Promote Skin Wound Healing by Activating PI3K/AKT Pathway through PTEN

• Xiu C
• Zheng H
• Jiang M
• Li J
• Zhou Y
• Mu L
• Liu W

• KMID: 2536206
• DOI: http://doi.org/10.15283/ijsc21135

Abstract

Background and Objectives
The goal of this study was to investigate the mechanism of mesenchymal stem cell (MSC)-derived microRNA (miR)-150-5p-expressing exosomes in promoting skin wound healing through activating PI3K/AKT pathway by PTEN.
Methods and Results
Human umbilical cord (HUC)-MSCs were infected with miR-150-5p overexpression and its con-trol lentivirus, and HUC-MSCs-derived exosomes (MSCs-Exos) with stable expression of miR-150-5p were obtained. HaCaT cells were induced by H₂O₂ to establish a cellular model of skin injury, in which the expression of miR-150-5p and PTEN and the phosphorylation of PI3K and AKT were evaluated. HaCaT cells were transfected with pcDNA3.1-PTEN or pcDNA3.1 and then cultured with normal exosomes or exosomes stably expressing miR-150-5p. Cell proliferation was inspected by CCK-8. Cell migration was detected by scratch test and cell apoptosis by flow cytometry. The starBase tool was used to predict the binding site of miR-150-5p to PTEN. Dual-luciferase reporter assay and RIP assay were applied to assess the interaction between miR-150-5p and PTEN. In H₂O₂ -induced HaCaT cells, the miR-150-5p expression decreased, and PTEN expression increased in a concentration-dependent manner. MSCs-Exos promoted the growth and migration of H₂O₂ -induced HaCaT cells and inhibited their apoptosis. In addition, overexpression of exosomal miR-150-5p enhanced the protective effect of MSCs-Exos on H₂O₂ -induced HaCaT cells; PTEN overexpression in HaCaT cells partially restrained miR-150-5p-mediated inhibition on H₂O₂ -induced injury in HaCaT cells. PTEN was a target gene of miR-150-5p. MiR-150-5p regulated PI3K/AKT pathway through PTEN.
Conclusions
MSCs-derived miR-150-5p-expressing exosomes promote skin wound healing by activating PI3K/AKT pathway through PTEN.

Keyword

Mesenchymal stem cell-derived exosomes; miR-150-5p; PTEN; PI3K/AKT pathway; Skin wound healing; Proliferation; Migration; Apoptosis

Figure

• 

  Fig. 1 Isolation and identification of the MSCs-Exos. (A) The morphology of primary MSCs under an inverted microscope. (B) Oil Red O staining after adipogenic differentiation. (C) Alizarin red staining after osteogenic differentiation. (D) Alcian blue staining detecting chondrogenic differen-tiation. (E) The expression of surface antigen markers for MSCs (CD29, CD90, CD44, and CD34) evaluated by flow cytometry assay. (F) Obser-vation of exosomes by TEM (scale bar: 200 nm). (G) Western blot analysis of the protein expression of exosome surface markers (CD63 and CD81), CANX, GM130, and Histone H3. MSC: mesenchymal stem cells, TEM: transmission electron microscopy, Exos: exosomes.

• 

  Fig. 2 MSCs-Exos promote the proliferation and migration and inhibit the apoptosis of H2O2-induced HaCaT cells. (A) Detection of the effects of H2O2 at different concentrations on the viability of HaCaT cells by CCK-8 method. (B) Detection of the apoptosis of HaCaT cells by flow cytometry. (C) Detection of the viability of HaCaT cells by the CCK-8 method. (D) Detection of the migration of HaCaT cells in each group by the plate scratch test. (E) Detection of the apoptosis of HaCaT cells by flow cytometry. *p<0.05, **p<0.01, ***p< 0.001, n=3. The data were presented in the form of mean±standard deviation. The comparison among multiple groups was performed by one-way analysis of variance. Tukey’s multiple tests were used for post hoc comparison. MSC: mesenchymal stem cell, Exos: exosomes.

• 

  Fig. 3 MiR-150-5p expressed in the MSCs-Exos effectively improves H2O2-treated HaCaT cell viability. (A) Detection of miR-150-5p expression in HaCaT cells treated with H2O2 by RT-qPCR. (B) Detection of miR-150-5p expression in MSC culture medium supernatants after exosome isolation and exosomes solution by RT-qPCR. (C) Detection of miR-150-5p expression in exosomes after digestion with RNase by RT-qPCR. (D) Fluorescent images of lentivirus-infected cells. (E) Verification of the overexpression and inhibition efficacy of miR-150-5p by RT-qPCR. (F) Detection of the viability of HaCaT cells in each group by the CCK-8 method. (G) Determination of the migration of HaCaT cells in each group by the plate scratch test. (H) Detection of the apoptosis of HaCaT cells in each group by flow cytometry. *p<0.05, **p<0.01, ***p<0.001, n=3. The data were presented in the form of mean±standard deviation. The comparison among multiple groups was performed by one-way analysis of variance. Tukey’s multiple tests were used for the post hoc comparison. MSC: mesenchymal stem cells, Exos: exosomes.

• 

  Fig. 4 MiR-150-5p regulates PI3K/AKT pathway by mediating PTEN. (A) Prediction of the binding site between miR-150-5p and PTEN by starBase (http://starbase.sysu.edu.cn/). (B, C) The detection of the expression level of PTEN in HaCaT cells treated with different concentrations of H2O2 by RT-qPCR (B) and western blot analysis (C). (D, E) Verification of the interaction between miR-150-5p and PTEN by dual-luciferase reporter assay (D) and RIP test (E). (F) The detections of miR-150-5p expression and PTEN mRNA expression in HaCaT cells transfected with miR-150-5p mimic, miR-150-5p inhibitor, or miR-150-5p inhibitor＋sh-PTEN by RT-qPCR. (G) PTEN, PI3K, and AKT protein expression and the phosphorylation of PI3K and AKT in HaCaT cells transfected with miR-150-5p mimic, miR-150-5p inhibitor, or miR-150-5p inhibitor＋sh-PTEN by western blot analysis. *p<0.05, **p<0.01, ***p<0.001, n=3. The data were presented in the form of mean±standard deviation. The comparison among multiple groups was performed by one-way analysis of variance. Tukey’s multiple tests were used for the post hoc comparison. MSC: mesenchymal stem cells, Exos: exosomes.

• 

  Fig. 5 PTEN overexpression restrains exosomal miR-150-5p-mediated inhibition on H2O2-induced injury in HaCaT cells. (A, B) The expression of PTEN in HaCaT cells transfected with pcDNA3.1 or pcDNA3.1-PTEN by RT-qPCR (A) and western blot analysis (B). (C, D) The expression of PTEN in HaCaT cells by RT-qPCR (C) and western blot analysis (D). (E) Detection of the viability of HaCaT cells by the CCK-8 method. (F) Detection of the migration of HaCaT cells in each group by the plate scratch test. (G) Detection of apoptosis of HaCaT cells in each group by flow cytometry. (H) Detection of the phosphorylation level of PI3K and AKT by western blot analysis. *p<0.05, **p<0.01, ***p<0.001, n=3. The data were presented in the form of mean±standard deviation. The comparison among multiple groups was performed by one-way analysis of variance. Tukey’s multiple tests were used for the post hoc comparison. MSC: mesenchymal stem cells, Exos: exosomes.

Reference

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