Transcription Factor NFAT5 Promotes Glioblastoma Cell-driven Angiogenesis via SBF2-AS1/miR-338-3p-Mediated EGFL7 Expression Change

Abstract

Glioblastoma (GBM) is the most aggressive primary intracranial tumor of adults and confers a poor prognosis due to high vascularization. Hence anti-angiogenic therapy has become a promising strategy for GBM treatment. In this study, the transcription factor nuclear factor of activated T-cells 5 (NFAT5) was significantly elevated in glioma samples and GBM cell lines, and positively correlated with glioma WHO grades. Knockdown of NFAT5 inhibited GBM cell-driven angiogenesis. Furthermore, long non-coding RNA SBF2 antisense RNA 1 (SBF2-AS1) was upregulated in glioma samples and knockdown of SBF2-AS1 impaired GBM-induced angiogenesis. Downregulation of NFAT5 decreased SBF2-AS1 expression at transcriptional level. In addition, knockdown of SBF2-AS1 repressed GBM cell-driven angiogenesis via enhancing the inhibitory effect of miR-338-3p on EGF like domain multiple 7 (EGFL7). In vivo study demonstrated that the combination of NFAT5 knockdown and SBF2-AS1 knockdown produced the smallest xenograft volume and the lowest microvessel density. NFAT5/SBF2-AS1/miR-338-3p/EGFL7 pathway may provide novel targets for glioma anti-angiogenic treatment.

Keywords: EGFL7; NFAT5; SBF2-AS1; glioblastoma angiogenesis; long non-coding RNA; miR-338-3p; transcription factor.

Figure 1

NFAT5 was upregulated in glioma tissues and GBM cell lines. (A) Representative patterns of NFAT5 expression levels that were determined by immunohistochemistry staining in glioma tissues and normal brain tissues. A1–A2 normal brain tissues (n = 5), B1–B2 grade I gliomas (n = 8), C1–C2 grade II gliomas (n = 10), D1–D2 grade III gliomas (n = 14), E1–E2 grade IV gliomas (n = 15). Original magnification, × 100 in A1–E1, × 400 in A2–E2. (B) Association of NFAT5 expression levels with WHO grade. P value was estimated by chi-square test. (C) Representative patterns of NFAT5 expression levels that were detected by Western blot in glioma tissues (Grades I–II, 7 cases; Grades III–IV, 12 cases) and NBTs (5 cases). Data represent mean ± s.d. (each case was determined by Western blot for three times), ^#P and ^*P < 0.05, ^##P < 0.01. (D) Expression of NFAT5 was detected by Western blot in normal human astrocytes, U87 and U118 cell lines. Data represent mean ± s.d. (n = 3, each). ^*P < 0.05.

Figure 2

Knockdown of NFAT5 suppressed GBM cell-driven angiogenesis in vitro. NFAT5 was knockdown in U87 and U118 cells. Effects of GBM cell-conditioned medium on ECs were detected. (A) ECs cell viability was measured by CCK-8 assay. (B) ECs migration was measured by Transwell assay. (C) ECs tube formation was measured by Matrigel tube formation assay. Data represent mean ± s.d. (n = 4, each). ^*P < 0.05, ^**P < 0.01. Scale bar represents 30 μm.

Figure 3

Downregulation of SBF2-AS1 inhibited GBM cell-driven angiogenesis in vitro. (A) Expression levels of SBF2-AS1 were detected by qRT-PCR in glioma tissues (Grades I–II, seven cases; Grade III, six cases; Grade IV, six cases) and NBTs (five cases). Data represent mean ± s.d. (n = 3, each), ^#P and ^*P < 0.05,^**P, ^&&P, and ^##P < 0.01. (B) Expression levels of SBF2-AS1 were detected by qRT-PCR in normal human astrocytes, U87 and U118 cell lines. Data represent mean ± s.d. (n = 4), ^**P < 0.01. SBF2-AS1 was knockdown in U87 and U118 cells. Effects of GBM cell-conditioned medium on ECs were detected. (C) ECs cell viability was measured by CCK-8 assay. (D) ECs migration was measured by Transwell assay. (E) ECs tube formation was measured by Matrigel tube formation assay. Data represent mean ± s.d. (n = 4, each). ^*P < 0.05, ^**P < 0.01. Scale bar represents 30 μm.

Figure 4

NFAT5 regulated SBF2-AS1 at transcriptional level. (A) Linear regression analysis was performed to each individual NFAT5 and SBF2-AS1 expression, P < 0.01. (B) NFAT5 was knockdown in U87 and U118 cells. SBF2-AS1 levels in U87 and U118 cells were measured by qRT-PCR. Data represent mean ± s.d. (n = 4, each). ^**P < 0.01. (C) Schematic representation of the human SBF2-AS1 promoter region. Chromatin immunoprecipitation PCR products for putative NFAT5-binding sites and an upstream region not expected to associate with NFAT5 are amplified by PCR using their specific primers (n = 3, each).

Figure 5

Combination of NFAT5 and SBF2-AS1 knockdown inhibited GBM cell-driven angiogenesis. NFAT5 and SBF2-AS1 were knockdown in U87 and U118 cells. Effects of NFAT5 and SBF2-AS1 on ECs cell viability (A), migration (B), and tube formation (C) were measured. Data represent mean ± s.d. (n = 4). ^**P < 0.01. Scale bar represents 30 μm.

Figure 6

miR-330-3p was sponged by SBF2-AS1 and was involved in SBF2-AS1-mediated GBM cell-driven angiogenesis. (A) Luciferase reporter vectors that carry cDNA sequences of SBF2-AS1 containing the wild-type or mutant miR-338-3p binding site and the seed region of miR-338-3p. (B) Relative luciferase activity in HEK293T cells which were co-transfected pmirGLO-SBF2-AS1-Wt or Mut and agomir-338-3p were determined. Data represent mean ± s.d. (n = 4, each). ^*P < 0.05. (C,D) RNA immunoprecipitation assay was performed with normal mouse IgG or anti-Ago2 in U87 and U118 cells. Relative enrichment of SBF2-AS1 and miR-338-3p were determined by qRT-PCR. Data represent mean ±s.d. (n = 4, each). ^#P < 0.05, ^##P, and ^**P < 0.01. Agomir-338-3p or antagomir-338-3p was transfected in cells that SBF2-AS1 was stably knocked down. Effects of co-transfection of SBF2-AS1 and miR-338-3p on ECs cell viability (E), migration (F), and tube formation (G) were measured. Data represent mean ± s.d. (n = 4, each), ^**P < 0.01. Scale bar represents 30 μm.

Figure 7

miR-338-3p downregulated EGFL7 by targeting its 3′-UTR. (A) The binding site on EGFL7 3′-UTR and the seed region of miR-338-3p. (B) Relative luciferase activity in HEK293T cells that co-transfected pmirGLO-EGFL7-3′-UTR-Wt or Muts and agomir-338-3p were determined. Data represent mean ± s.d. (n = 3, each). ^**P < 0.01. Agomir-338-3p was transiently transfected in U87 and U118 cells that were stably overexpressed EGFL7 with or without 3′-UTR. Effects of co-transfection of agomir-338-3p and EGFL7 with or without 3′-UTR on EGFL7 protein level (C), secretion (D) were measured. Effect of miR-338-3p on EGFL7 mRNA level (E) was measured. Data represent mean ± s.d. (n = 4). ^*P and ^#P < 0.05, ^&&P < 0.01.

Figure 8

miR-338-3p suppressed EGFL7-induced pro-angiogenic effect by targeting EGFL7 3′-UTR. (A) Effects of co-transfection of agomir-338-3p and EGFL7 with or without 3′-UTR on ECs cell viability (B), migration (C), and tube formation (D) were measured. Effect of co-transfection of agomir-338-3p and EGFL7 with or without 3′-UTR on ERK levels was measured by Western blot. Data represent mean ± s.d. (n = 4). ^*P and ^#P < 0.05, ^&&P < 0.01. Scale bar represents 30 μm.

Figure 9

Knockdown of NFAT5 and SBF2-AS1 inhibited EGFL7 protein level and secretion without affecting EGFL7 mRNA level. Effects of NFAT5 knockdown on EGFL7 protein level (A), secretion (B), and mRNA level (C). Effects of SBF2-AS1 knockdown on EGFL7 protein level (D), secretion (E), and mRNA level (F). Effects of combination knockdown of NFAT5 and SBF2-AS1 on EGFL7 protein level (G), secretion (H), and mRNA level (I). Data represent mean ± s.d. (n = 4). ^*P < 0.05, ^**P < 0.01.

Figure 10

Combination of NFAT5 knockdown and SBF2-AS1 knockdown inhibited xenograft glioma growth and the microvessel density in vivo. The stable expressing U87 and U118 cells were used for the in vivo study. (A) The nude mice carrying tumors from respective groups and a sample tumor from each group were shown. (B) Xenograft glioma growth curves in nude mice. Tumor volume was measured every 5 days after injection. Data represent mean ± s.d. (n = 7, each group). (C) Tumors were harvested on day 45 and paraffin-embedded tumor sections were prepared. CD-31 immunohistochemistry staining was performed to determine the microvessel density (MVD). (magnification, × 400; scale bar = 50 μm). Data represent mean ± s.d. (n = 4, each group). ^*P, ^&P, and ^#P < 0.05, ^&&P and ^**P < 0.01.

Figure 11

The Schematic representation of NFAT5/SBF2-AS1/miR-338-3p/EGFL7 axis in GBM cell-driven angiogenesis.