Glioma cells enhance angiogenesis and inhibit endothelial cell apoptosis through the release of exosomes that contain long non-coding RNA CCAT2

Abstract

Angiogenesis is a key event in the progression of gliomas. Exosomes, as signaling extracellular organelles, modulate the tumor microenvironment and promote angiogenesis and tumor progression. We previously demonstrated that long intergenic non-coding RNA CCAT2 (linc-CCAT2) was overexpressed in glioma tissues and functioned to promote glioma progression. Therefore, this study aimed to explore an underlying mechanism of glioma cell-affected angiogenesis. First, qRT-PCR was used to determine the expression level of linc-CCAT2 in 4 glioma cell lines and 293T cells, and the results revealed that the U87-MG cells exhibited the highest expression level. Subsequently, the pro-angiogenesis function of exosomes that were derived from negative control shRNA-treated U87-MG cells (ncU87-Exo) and linc-CCAT2 shRNA-treated U87-MG cells (shU87-Exo) was evaluated in vitro and in vivo. We found that ncU87-Exo, which was enriched in linc-CCAT2, could be taken up by HUVECs. ncU87-Exo improved the linc-CCAT2 expression level in HUVECs and more strongly promoted HUVEC migration, proliferation, tubular-like structure formation in vitro and arteriole formation in vivo as well as inhibited HUVEC apoptosis induced by hypoxia. Further mechanistic studies revealed that ncU87-Exo could upregulate VEGFA and TGFβ expression in HUVECs as well as promote Bcl-2 expression and inhibit Bax and caspase-3 expression. Finally, gain-/loss-of-function studies revealed that the overexpression of linc-CCAT2 in HUVECs activated VEGFA and TGFβ, promoted angiogenesis, promoted Bcl-2 expression and inhibited Bax and caspase-3 expression, thus decreasing apoptosis. Downregulation of linc-CCAT2 revealed the opposite effect. Thus, our results revealed a new exosome‑mediated mechanism by which glioma cells could promote angiogenesis through the transfer of linc-CCAT2 by exosomes to endothelial cells. Moreover, we suggest that exosomes and linc-CCAT2 are putative therapeutic targets in glioma.

Figure 1.

Characterization of exosomes released by ncU87-MG cells and shU87-MG cells. (A) linc-CCAT2 expression levels were evaluated by qRT-PCR in the following four glioma cell lines: U87-MG, U251, A172, and T98G; non-glioma 293T cells were used as controls. U87-MG cells exhibited the highest expression level of linc-CCAT2 (compared with the 293T cells, *P<0.01; compared with the U251, A172, and T98G cells, ^#P<0.05). (B) The knockdown effects of linc-CCAT2 were assessed by qRT-PCR in U87-MG cells transfected with the shRNA or negative controls; the linc-CCAT2 shRNA3 was the most efficient in silencing linc-CCAT2 mRNA (compared with the normal and negative controls, *P<0.01; compared with shRNA1 and shRNA2, ^#P<0.05). (C) ncU87-Exo cells were highly enriched in linc-CCAT2 transcripts compared with shU87-Exo (*P<0.01). (D and E) The nanoparticle size distribution and concentrations for ncU87-Exo and shU87-Exo were obtained via NTA, and the morphology of ncU87-Exo and shU87-Exo was observed by TEM. (F) The ncU87-Exo and shU87-Exo concentrations were normalized to final cell counts; there were no significant differences observed between ncU87-Exo and shU87-Exo cells (P>0.05). (G) Western blot analysis of exosomal markers Alix, Tsg101, and CD9 in ncU87-Exo and shU87-Exo; equal amounts of exosomes (300 ng) were used for the assay. qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; NTA, Nanoparticle Tracking Analysis; TEM, transmission electron microscopy.

Figure 2.

ncU87-Exo and shU87-Exo are internalized by HUVECs. (A) Immunofluorescence images of DAPI (blue)-CD31 (red) HBMECs co-cultured with CM-Dio (green) labeled ncU87-Exo and shU87-Exo at 6 h. (B) qRT-PCR was applied to determine linc-CCAT2 expression levels in HUVECs when incubated with 100 µg/ml ncU87-Exo and shU87-Exo for 24 h; the linc-CCAT2 expression level in ncU87-Exo-treated HUVECs was significantly higher than that in the shU87-Exo-treated HUVECs (*P<0.01). HUVECs, human umbilical vein endothelial cells; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction.

Figure 3.

ncU87-Exo and shU87-Exo regulates HUVEC migration, proliferation, and tube formation in vitro and angiogenesis in vivo. (A and B) The migration ability was assessed with a scratch-wound assay, and both ncU87-Exo and shU87-Exo significantly enhanced the motility of HUVECs, while ncU87-Exo exhibited a more efficient pro-migration ability. (C) The proliferation was assessed with the CCK-8 assay; ncU87-Exo significantly stimulated HUVEC proliferation, while shU87-Exo only slightly promoted HUVEC proliferation. (D-G) The tube formation assay was performed on growth factor-reduced Matrigel. The number of total branching points, total tube length, and total loops at 6 h in the ncU87-Exo-treated HUVECs was higher than that in the shU87-Exo-treated HUVECs. (H and I) Similar to the in vitro experiment, ncU87-Exo induced CAM arteriole formation to a greater extent, in contrast to shU87-Exo. Representative images of the CAM assays are presented in the left panel, and the number of vessels is presented in the right panel (n=3). (*P<0.05 when compared to the control medium; ^#P<0.05 when compared to shU87-Exo). HUVECs, human umbilical vein endothelial cells; CCK-8, Cell Counting Kit-8 assay.

Figure 4.

ncU87-Exo and shU87-Exo regulate angiogenesis related-genes and protein expression in HUVECs. (A) qRT-PCR analysis of the expression level of angiogenesis-related genes in HUVECs treated by ncU87-Exo and shU87-Exo. Compared with the shU87-Exo group, ncU87-Exo significantly upregulated VEGF, TGFβ, FGF and KDR gene expression. (B) ELISA analysis of the secretion level of angiogenesis-related proteins in HUVECs treated by ncU87-Exo and shU87-Exo. Compared with the shU87-Exo group, ncU87-Exo significantly increased VEGF, TGFβ, and FGF protein secretion. (*P<0.05 when compared to the control medium; ^#P<0.05 when compared to shU87-Exo). HUVECs, human umbilical vein endothelial cells; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; VEGF, vascular endothelial growth factor; TGFβ, transforming growth factor β.

Figure 5.

ncU87-Exo and shU87-Exo decrease HUVEC apoptosis induced by hypoxia in vitro. Both ncU87-Exo and shU87-Exo decreased apoptosis of HUVECs induced by hypoxia, and ncU87-Exo exhibited more efficient anti-apoptosis. (A) Representative FCM images of PI and Annexin V-FITC double-stained HUVEC apoptosis. (B) The bar chart of PI and Annexin V-FITC double-stained HUVEC apoptosis. (*P<0.01, hypoxia (−) group vs. hypoxia (+) control medium, ncU87-Exo and shU87-Exo group; ^#P<0.05, hypoxia (+) ncU87-Exo and shU87-Exo group vs. hypoxia (+) control medium; ^&P<0.05, hypoxia (+) ncU87-Exo group vs. hypoxia (+) shU87-Exo group). HUVECs, human umbilical vein endothelial cells.

Figure 6.

ncU87-Exo and shU87-Exo regulate the expression of apoptosis-related factors in HUVECs induced by hypoxia. (A) qRT-PCR analysis of the expression level of apoptosis-related factors Bcl-2, Bax, and caspase-3 in HUVECs treated by ncU87-Exo and shU87-Exo after hypoxia. Compared with the control medium group and the shU87-Exo group, ncU87-Exo significantly upregulated Bcl-2 gene expression and inhibited Bax and caspase-3 gene expression. (B and C) Western blot analysis revealed that both ncU87-Exo and shU87-Exo increased Bcl-2 expression and decreased Bax and caspase-3 expression, while ncU87-Exo was more efficient. (*P<0.05 when compared to the control medium; ^#P<0.05 when compared to shU87-Exo). HUVECs, human umbilical vein endothelial cells; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; Bcl-2, B-cell leukemia 2; Bax, Bcl-2 associated X protein.

Figure 7.

linc-CCAT2 regulates HUVEC migration, proliferation, and tube formation in vitro. (A) The linc-CCAT2 expression levels were evaluated by qRT-PCR in linc-CCAT2 overexpression, downregulation, negative control, and normal HUVECs. linc-CCAT2 overexpression in HUVECs exhibited the highest expression level of linc-CCAT2, while downregulation of linc-CCAT2 in HUVECs exhibited the lowest expression level of linc-CCAT2. (B and C) The migration ability was assessed with the scratch-wound assay; linc-CCAT2 overexpression increased HUVEC migration, while downregulation of linc-CCAT2 significantly decreased HUVEC migration. (D) The proliferation was assessed with the CCK-8 assay; linc-CCAT2 overexpression promoted HUVEC proliferation, while linc-CCAT2 downexpression inhibited HUVEC proliferation. (E-H) The tube formation assay was performed on growth factor-reduced Matrigel. The total number of branching points, total tube length, and total loops at 6 h in the linc-CCAT2 overexpression in HUVECs was higher than in the negative control HUVECs, while downregulation of linc-CCAT2 in HUVECs was lower than the negative control HUVECs. (*P<0.05 when compared to the negative control; ^#P<0.05 when compared to the negative control). HUVECs, human umbilical vein endothelial cells; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; CCK-8, Cell Counting Kit-8 assay.

Figure 8.

linc-CCAT2 regulates angiogenesis-related genes and protein expression in HUVECs. (A) qRT-PCR analysis of the expression level of angiogenesis-related genes in linc-CCAT2 overexpression, downregulation and negative control HUVECs. linc-CCAT2 overexpression in HUVECs increased the gene expression of VEGF, TGFβ, and KDR, while downregulation of linc-CCAT2 inhibited VEGF, TGFβ, and KDR expression. (B) ELISA analysis of the level of secreted angiogenesis-related proteins in linc-CCAT2 overexpression, downregulation and negative control HUVECs. linc-CCAT2 overexpression in HUVECs increased VEGF and TGFβ protein secretion, while downregulation of linc-CCAT2 inhibited VEGF and TGFβ protein secretion. (*P<0.05 when compared to the negative control; ^#P<0.05 when compared to the negative control). HUVECs, human umbilical vein endothelial cells; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; VEGF, vascular endothelial growth factor; TGFβ, transforming growth factor β.

Figure 9.

linc-CCAT2 decreases HUVEC apoptosis induced by hypoxia in vitro. (A) Representative FCM images of PI and Annexin V-FITC double-stained HUVEC apoptosis. (B) The bar chart of PI and Annexin V-FITC double-stained HUVEC apoptosis. The apoptosis percentage in the downregulated lin-CCAT2 HUVECs was significantly higher than that in the negative control HUVECs, while the apoptosis percentage in the overexpressed linc-CCAT2 HUVECs was markedly lower than that in the negative control HUVECs. (*P<0.05 when compared to negative control; ^#P<0.05 when compared to negative control). HUVECs, human umbilical vein endothelial cells.

Figure 10.

linc-CCAT2 regulates apoptosis-related factor expression in HUVECs induced by hypoxia. (A) qRT-PCR analysis of the expression level of apoptosis-related factors Bcl-2, Bax and caspase-3 in linc-CCAT2 overexpression, downregulation and negative control HUVECs. Compared with the negative control HUVECs, overexpression promoted Bcl-2 gene expression and inhibited Bax and caspase-3 gene expression, while downregulation of linc-CCAT2 in HUVECs inhibited Bcl-2 gene expression and promoted Bax and caspase-3 gene expression. (B and C) Western blot analysis of the expression level of Bcl-2, Bax, and caspase-3 in HUVECs. Compared with the negative control HUVECs, linc-CCAT2 overexpression improved Bcl-2 protein expression and decreased Bax and caspase-3 protein expression, while downregulation of linc-CCAT2 decreased Bcl-2 protein expression and increased Bax and caspase-3 protein expression. (*P<0.05 when compared to the negative control; ^#P<0.05 when compared to the negative control). HUVECs, human umbilical vein endothelial cells; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; Bcl-2, B-cell leukemia 2; Bax, Bcl-2 associated X protein.