Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Aug;38(2):785-798.
doi: 10.3892/or.2017.5742. Epub 2017 Jun 22.

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

Hai-Li Lang  1 , Guo-Wen Hu  2 , Bo Zhang  3 , Wei Kuang  2 , Yong Chen  1 , Lei Wu  2 , Guo-Hai Xu  1
Affiliations

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

Hai-Li Lang et al. Oncol Rep. 2017 Aug.

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.

PubMed Disclaimer

Figures

Figure 1.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Carmeliet P. Angiogenesis in life, disease and medicine. Nature. 2005;438:932–936. doi: 10.1038/nature04478. - DOI - PubMed
    1. Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature. 2011;473:298–307. doi: 10.1038/nature10144. - DOI - PMC - PubMed
    1. Kerbel RS. Tumor angiogenesis. N Engl J Med. 2008;358:2039–2049. doi: 10.1056/NEJMra0706596. - DOI - PMC - PubMed
    1. Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst. 1990;82:4–6. doi: 10.1093/jnci/82.1.4. - DOI - PubMed
    1. Millauer B, Shawver LK, Plate KH, Risau W, Ullrich A. Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature. 1994;367:576–579. doi: 10.1038/367576a0. - DOI - PubMed

MeSH terms

Substances