Upregulation of miR-107 Inhibits Glioma Angiogenesis and VEGF Expression

Abstract

MicroRNAs can function as oncogenes or tumor suppressors in glioma. Previously, we showed that miR-107 inhibits glioma cell proliferation, migration, and invasion. Since tumor growth and invasion are closely related to angiogenesis, we further examined the role of miR-107 in glioma angiogenesis. In a co-culture of glioma cells and human brain microvascular endothelial cells (HBMVEC), overexpression of miR-107 in glioma cells led to the inhibition of HBMVEC proliferation, migration, and tube formation ability. ELISA, RT-PCR, and western blot assays revealed that upregulation of miR-107 in glioma cells inhibits VEGF expression. Our findings collectively support the critical involvement of miR-107 in glioma cell angiogenesis and highlight its potential as a therapeutic target for glioma.

Keywords: Angiogenesis; Glioma; HBMVEC; VEGF; miR-107.

Fig. 1

Upregulation of miR-107 inhibits glioma growth and angiogenesis in vivo. a Nude mice were subcutaneously inoculated with U87 cells transduced with GFP or miR-107, and neoplasms obtained 6 weeks later. Representative images are shown. b Comparison of tumor volumes between miR-107 and GFP groups every 7 days. c Quantification tumor weight after nude mice were subcutaneously inoculated with U87 cells transduced with GFP or miR-107. d Representative image of a neoplasm section stained for CD31 to determine vascular formation (×400 magnification). Scale bars 50 μm. e CD31-positive microvessels were counted in three different fields per section at ×400 magnification. *P < 0.05. GFP transduced with GFP only, miR-107 transduced with GFP and miR-107

Fig. 2

Overexpression of miR-107 in glioma cells inhibits HBMVEC proliferation and migration in vitro. a U87 and A172 cells (green fluorescence, transduced with GFP or miR-107) were co-cultured with HBMVECs (red fluorescence, stained with PKH26) in six-well plates. Representative images, obtained 24 h later, are shown (×200 magnification). Scale bars 100 μm. b The effect of supernatant derived from U87 and A172 cells transduced with GFP or miR-107 on HBMVECs proliferation. Representative images are shown. Scale bars 100 μm. c Representative fluorescence microscopy image (×100 magnification) showing the effects of the supernatant fractions of U87 and A172 cells (transduced with GFP or miR-107) on HBMVEC (red fluorescence, stained with PKH26) migration in the wound-healing assay. Images were acquired directly after scratching (t = 0) and 24 h later (t = 24). Scale bars 200 μm. d The numbers of HBMVECs co-cultured with U87 and A172 glioma cells (transduced with GFP or miR-107) were calculated using ImageJ software. e Quantitation of the number of HBMVECs after treated with supernatant derived from U87 and A172 cells transduced with GFP or miR-107 using ImageJ software. f Quantitation of HBMVEC migration to the scratch using ImageJ software. *P < 0.05. GFP transduced with GFP only, miR-107 transduced with GFP and miR-107

Fig. 3

Upregulation of miR-107 in glioma cells suppresses tubule formation in the HBMVEC and glioma cell co-culture system. a, b U87 or A172 glioma cells (green fluorescence, transduced with GFP or miR-107) and HBMVECs (red fluorescence, stained with PKH26) were co-cultured on Matrigel-coated plates, and tubule formation assessed after 48 h. Representative images are shown (×100). Scale bars 150 μm. c ImageJ software was applied to evaluate tubule crosses in a, b. *P < 0.05. GFP transduced with GFP only, miR-107 transduced with GFP and miR-107

Fig. 4

Overexpression of miR-107 inhibits glioma VEGF expression. a ELISA assay data revealed that compared with the GFP group, VEGF expression levels are inhibited in the supernatant fractions of glioma cell lines, U87 and A172, transduced with miR-107. b RT-PCR disclosed downregulation of VEGF mRNA in U87 and A172 glioma cells transduced with miR-107. c Western blot results showed that miR-107 inhibits VEGF protein expression in U87 and A172 glioma cells. d Western blot assay showed that VEGF protein expression was downregulated in neoplasm treated with miR-107.*P < 0.05. GFP transduced with GFP only, miR-107 transduced with GFP and miR-107