Exosomes derived from stem cells of human deciduous exfoliated teeth inhibit angiogenesis in vivo and in vitro via the transfer of miR-100-5p and miR-1246

Abstract

Background Anti-angiogenic therapy has been shown to be a promising strategy for anti-tumor treatment. Increasing evidence indicates that tumor angiogenesis is affected by exosomes that are secreted by mesenchymal stem cells (MSCs), but whether exosomes derived from MSCs suppress or promote angiogenesis remain paradoxical. The purpose of this study focused on understanding the potential role of exosomes derived from stem cells of human deciduous exfoliated teeth (SHED-Exos) in regulating angiogenesis and the underlying molecular mechanism. Methods Exosomes were isolated from supernatants of SHED cells using an exosome purification kit and were characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. Cell Counting Kit-8, flow cytometric assays, western blots, wound healing and transwell migration assays were performed to characterize the roles of SHED-Exos on cell proliferation, apoptosis and migration of human umbilical vein endothelial cells (HUVECs). The anti-angiogenic activity of SHED-Exos was assessed via a tube formation assay of endothelial cells and angiogenesis-related factors were analyzed by western blotting. In vivo, we used the chick chorioallantoic membrane (CAM) assay and an oral squamous cell carcinoma (OSCC) xenograft transplantation model with nude mice that received multi-point injections at three-day intervals to evaluate the effects on angiogenesis. Furthermore, the sequencing of microRNAs (miRNAs) in SHED-Exos was performed to investigate the underlying anti-angiogenic mechanism. Results The results showed that SHED-Exos inhibit cell proliferation and migration and induce apoptosis in HUVECs. SHED-Exos suppress the tube-like structure formation of HUVECs in vitro. SHED-Exos downregulate several angiogenesis-related factors, including VEGFA, MMP-9 and ANGPT1. In vivo, the chick CAM assay verified that treatment with SHED-Exos inhibits micro-vascular formation, and importantly, significantly reduces the micro-vascular formation of tumors generated from xenografted OSCC cells, which was associated with the inhibition of tumor growth in vivo. Mechanistically, our data suggested that SHED-Exos are enriched with miR-100-5p and miR-1246 and are transferred to endothelial cells, which results in decreased tube formation via the down-regulation of VEGFA expression. Conclusions These results demonstrate that SHED-Exos inhibit angiogenesis in vitro and in vivo, which suggests that SHED-Exos could potentially serve as a novel and effective therapeutic approach for anti-angiogenic treatment.

Phenformin suppresses angiogenesis through the regulation of exosomal microRNA-1246 and microRNA-205 levels derived from oral squamous cell carcinoma cells

“…Since we observed a high variation in the down-regulated miRNAs among the 3 replicate Phen-Exo samples ( Figure 5C ), we decided to investigate the 12 up-regulated miRNAs. We reviewed the literature to study which miRNA(s) among those 12 miRNAs is related to angiogenesis and found that miR-1246 has recently been shown to be an exosome component that suppresses the angiogenic function of endothelial cells in vitro and in vivo ( 26 ). Further, miR-205 has also been reported to be involved in angiogenesis ( 27 , 28 ), and therefore both miR-1246 and miR-205 (red arrows in Figure 5C ) were selected for further validation.…”

“…Next, we investigated how those two miRNAs regulate angiogenesis. Previous reports showed that miR-1246 and miR-205 can regulate angiogenesis through VEGFA ( 26 , 28 , 29 ), which is the most well-known member of the VEGF family and is a crucial modulator of vascular permeability and angiogenesis ( 30 , 31 ). Changes of VEGFA expression in the transfected HUVECs were detected both by qRT-PCR analysis ( Figures 6E, G ) and by Western blot analysis ( Figures 6F, H , Supplementary Figure 6 ).…”

“…The miR-205 has been reported to directly bind the 3’ UTR region of VEGFA mRNA to negatively control its expression in several cell types, such as breast cancer, colorectal cancer and bladder cancer as well as endothelial cells ( 44 – 47 ). For miR-1246, our previous study together other publications have already showed that miR-1246 didn’t directly bind VEGFA mRNA, but can indirectly target VEGF through miR-1246/ACE signal pathways to inhibit VEGF expression resulting in suppressing angiogenesis of endothelial cells ( 26 , 29 , 48 ). In the present study, we showed both VEGFA mRNA and protein expression levels were significantly decreased in both the miR-1246-mimics and the miR-205-mimics group, while opposite results were seen in the corresponding inhibitor groups.…”

Stem cell-derived exosomal transcriptomes for wound healing

“…Although most stem cell-exosomes derived miRNAs can promote cell proliferation, migration, and angiogenesis, a small proportion of miRNAs play a part in inhibiting wound healing. Exosomes enriched with miR-100-5p and miR-1246 from human deciduous tooth exfoliated dental stem cells were found to decrease cell proliferation and migration of HUVECs, induce cell apoptosis, and suppress tube like structure formation of HUVECs by downregulating several angiogenesis related factors, including VEGFA, MMP-9 and angiopoietin 1 (80), suggesting that miR-100-5p and miR-1246 play an anti-angiogenic role in wound healing.…”

The Effects of Mesenchymal Stem Cells on Oral Cancer and Possible Therapy Regime

“…Anti-angiogenic therapy may be a possible treatment strategy of tumors in the future. Liu et al (2022) treated TSCC CAL27 cells with exosomes derived from human deciduous exfoliated teeth by direct multi-point intratumor injection and found that the tumor volume was significantly smaller than that of the control group. Further experiments showed that exosomes from human deciduous exfoliated teeth downregulated VEGF-A expression through miR-100-5p and miR-1246, which significantly reduced the generation of microvasculature around TSCC.…”