Human umbilical cord-mesenchymal stem cells-derived exosomes carrying microRNA-15a-5p possess therapeutic effects on Wilms tumor via regulating septin 2

Abstract

The exact mechanism of miR-15a-5p shuttled by human umbilical cord-mesenchymal stem cell-derived exosomes (hUC-MSCs-Exo) in Wilms tumor (WT) was estimated. WT tissues were collected clinically. miR-15a-5p and septin 2 (SEPT2) expression levels were examined in tissues . hUC-MSCs-Exo were transfected with miR-15a-5p-related oligonucleotides and co-cultured with WT cells (G-401). In addition, SEPT2 loss-of-function was performed in G-401 cells. The biological functions of G-401 cells after treatments were evaluated. Moreover, tumor formation tests further assessed the role of exosomal miR-15a-5p in WT. The miR-15a-5p level was lower and the SEPT2 level was higher in WT. hUC-MSCs-Exo impaired the biological functions of G-401 cells. hUC-MSCs-Exo carried upregulated miR-15a-5p into G-401 cells, thereby lessening the tumorigenic properties of G-401 cells. Inhibition of SEPT2 suppressed the biological function of WT cells and upregulated SEPT2 reversed hUC-MSCs-Exo-mediated inhibition of G-401 cell growth. The tumorigenicity of G-401 cells in mice was impaired by hUC-MSCs-Exo overexpressing miR-15a-5p. The data prove that miR-15a-5p shuttled by hUC-MSCs-Exo negatively regulates SEPT2 expression, and disrupts WT cell growth in vivo and in vitro.

Keywords: MicroRNA-15a-5p; Wilms tumor; human umbilical cord-mesenchymal stem cells-derived exosomes; septin 2.

Graphical abstract

Figure 1.

miR-15a-5p is downregulated in WT. A. RT-qPCR tested miR-15a-5p expression in WT tissues and normal tissues; B. RT-qPCR tested miR-15a-5p expression in normal renal tubular epithelial HK-2 cell line and human WT G-401 cells; C. Microscopic observation of hUC-MSCs, and staining of adipocytes and osteoblasts; D. Flow cytometry tested hUC-MSCs surface markers; E. TEM observation of hUC-MSCs-Exo; F. NTA detection of hUC-MSCs-Exo; G. Western blot tested HSP70, CD81, CD9 and GRP94 in hUC-MSCs-Exo; H. Internalization of PKH26-labeled exosomes (red) in G-401 cells; I. RT-qPCR tested miR-15a-5p expression in hUC-MSCs-Exo. Measurement data were shown by the mean ± standard deviation.

Figure 2.

MSC-Exo impair the biological functions of G-401 cells. A. RT-qPCR tested miR-15a-5p in G-401 cells after co-culture with hUC-MSCs-EV; B. CCK-8 tested G-401 cell proliferation; C. Colony formation assay tested G-401 cell colony formation ability; D. Transwell assay tested G-401 cell migration; E. Transwell assay tested G-401 cell invasion; F. Flow cytometry tested G-401 cell apoptosis. Measurement data were shown by the mean ± standard deviation, * P < 0.05 vs the PBS group.

Figure 3.

MSC-Exo carrying miR-15a-5p lessen the tumorigenic properties of G-401 cells. A. RT-qPCR tested miR-15a-5p in G-401 cells after co-culture with hUC-MSCs-Exo; B. CCK-8 tested G-401 cell proliferation; C. Colony formation assay tested G-401 cell colony formation ability; D. Transwell assay tested G-401 cell migration; E. Transwell assay tested G-401 cell invasion; F. Flow cytometry tested G-401 cell apoptosis. Measurement data were shown by the mean ± standard deviation, * P < 0.05 vs the Exo-NC mimic group; # P < 0.05 vs the Exo-NC inhibitor group.

Figure 4.

miR-15a-5p targets SEPT2. A-B. RT-qPCR and Western blot tested SEPT2 expression in tissues and cells; C. Targeting site between miR-15a-5p and SEPT2; D. Dual luciferase reporter gene assay tested the targeting of miR-15a-5p and SEPT2; E. Western blot tested SEPT2 expression in G-401 cells after co-culture with hUC-MSCs-Exo; F. Pearson analyzed the correlation between miR-15a-5p and SEPT2. Measurement data were shown by the mean ± standard deviation, # P < 0.05 vs the PBS group; * P < 0.05 vs the Exo-NC mimic group; $ P < 0.05 vs the Exo-NC inhibitor group.

Figure 5.

Inhibition of SEPT2 represses the biological function of WT cells. A. Western blot tested SEPT2 expression in G-401 cells transfected with sh-SEPT2; B. CCK-8 tested G-401 cell proliferation; C. Colony formation assay tested G-401 cell colony formation ability; D. Transwell assay tested G-401 cell migration; E. Transwell assay tested G-401 cell invasion; F. Flow cytometry tested G-401 cell apoptosis. Measurement data were shown by the mean ± standard deviation, * P < 0.05 vs the sh-NC group.

Figure 6.

Upregulated SEPT2 reverses hUC-MSCs-Exo-mediated inhibition on G-401 cell growth. A. Western blot tested SEPT2 expression after co-culture of G-401 cells with hUC-MSCs-Exo; B. CCK-8 tested G-401 cell proliferation; C. Colony formation assay tested G-401 cell colony formation ability; D. Transwell assay tested G-401 cell migration; E. Transwell assay tested G-401 cell invasion; F. Flow cytometry tested G-401 cell apoptosis. Measurement data were shown by the mean ± standard deviation, * P < 0.05 vs the Exo+oe-NC group.

Figure 7.

MSC-EV carrying miR-15a-5p weakens tumorigenicity of G-401 cells in vivo. A. Tumor volume change in nude mice; B. Tumors and tumor weight. Measurement data were shown by the mean ± standard deviation, * P < 0.05 vs the PBS group; # P < 0.05 vs the Exo group.