ZRANB2/SNHG20/FOXK1 Axis regulates Vasculogenic mimicry formation in glioma

Abstract

Background: Glioma is the most common intracranial neoplasm with vasculogenic mimicry formation as one form of blood supply. Many RNA-binding proteins and long non-coding RNAs are involved in tumorigenesis of glioma.

Methods: The expression of ZRANB2, SNHG20 and FOXK1 in glioma were detected by real-time PCR or western blot. The function of ZRANB2/SNHG20/FOXK1 axis in glioma associated with vasculogenic mimicry formation was analyzed.

Results: ZRANB2 is up-regulated in glioma tissues and glioma cells. ZRANB2 knockdown inhibits the proliferation, migration, invasion and vasculogenic mimicry formation of glioma cells. ZRANB2 binds to SNHG20 and increases its stability. Knockdown of SNHG20 reduces the degradation of FOXK1 mRNA by SMD pathway. FOXK1 inhibits transcription by binding to the promoters of MMP1, MMP9 and VE-Cadherin and inhibits vasculogenic mimicry formation of glioma cells.

Conclusions: ZRANB2/SNHG20/FOXK1 axis plays an important role in regulating vasculogenic mimicry formation of glioma, which might provide new targets of glioma therapy.

Keywords: FOXK1; Glioma; SNHG20; Vasculogenic mimicry formation; ZRANB2.

Fig. 1

Endogenous expression of ZRANB2 and effect of ZRANB2 on biological behaviors of glioma cells. a Protein levels of ZRANB2 in NBTs, low-grade human glioma tissues (LGGTs) and high-grade human glioma tissues (HGGTs). Representative protein expressions and corresponding IDVs of ZRANB2 in NBTs (n = 15), low-grade glioma (n = 15), and high-grade glioma (n = 15) are shown; data are presented as mean ± SD.^**P < 0.01 vs. NBTs group, ^##P < 0.01 vs. LGGTs group. b Protein levels of ZRANB2 in NHA, U87 and U251 cells. Representative protein expressions and corresponding IDVs of ZRANB2 in NHA, U87 and U251 are shown; data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. NHA group. c Protein levels of MMP1, MMP9 and VE-Cadherin regulated by ZRANB2 in U87 and U251 cells. Representative protein expressions and corresponding IDVs of MMP1, MMP9, VE-Cadherin in U87 and U251 are shown; data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. ZRANB2(−)-NC group, ^**P < 0.01 vs. ZRANB2(−)-NC group, ^#P < 0.05 vs. ZRANB2(+)-NC group, ^##P < 0.01 vs. ZRANB2(+)-NC group. d CCK-8 assay was used to measure the effect of proliferation on U87 and U251 cells by ZRANB2. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. ZRANB2(−)-NC group, ^#P < 0.05 vs. ZRANB2(+)-NC group. e Transwell assay was used to measure the effect of migration and invasion on U87 and U251 cells by ZRANB2. Representative images and corresponding statistical plots are shown. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. ZRANB2 (−)-NC group, ^**P < 0.01 vs. ZRANB2(−)-NC group, ^#P < 0.05 vs. ZRANB2(+)-NC group, ^##P < 0.01 vs. ZRANB2(+)-NC group. Scale bars indicate 50 μm. f Three-dimensional culture was used to measure the effect of VM on U87 and U251 cells by ZRANB2. Representative images and corresponding statistical plots are shown. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. ZRANB2(−)-NC group, ^**P < 0.01 vs. ZRANB2(−)-NC group, ^#P < 0.05 vs. ZRANB2(+)-NC group. Scale bars indicate 50 μm

Fig. 2

Endogenous expression of SNHG20 and effect of SNHG20 on the biological behaviors of glioma cells. a Expression levels of SNHG20 in NBTs, LGGTs and HGGTs. Data are presented as mean ± SD (NBTs (n = 15), LGGTs (n = 15), HGGTs (n = 15)). ^**P < 0.01 vs. NBTs group, ^##P < 0.01 vs. LGGTs group. b Expression of SNHG20 in NHA, U87 and U251 cells. Data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. NHA group. c Expression levels of SNHG20 regulated by ZRANB2 in U87 and U251 cells. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. ZRANB2(−)-NC group, ^#P < 0.05 vs. ZRANB2(+)-NC group. d Protein levels of MMP1, MMP9 and VE-Cadherin regulated by SNHG20 in U87 and U251 cells. Representative protein expressions and corresponding IDVs of MMP1, MMP9, VE-Cadherin in U87 and U251 are shown; data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. SNHG20(−)-NC group, ^**P < 0.01 vs. SNHG20(−)-NC group, ^#P < 0.05 vs. SNHG20(+)-NC group, ^##P < 0.01 vs. SNHG20(+)-NC group. e CCK-8 assay was used to measure the effect of proliferation on U87 and U251 cells by SNHG20. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. SNHG20(−)-NC group, ^**P < 0.01 vs. SNHG20(−)-NC group, ^#P < 0.05 vs. SNHG20(+)-NC group, ^##P < 0.01 vs. SNHG20(+)-NC group. f Transwell assay was used to measure the effect of migration and invasion on U87 and U251 cells by SNHG20. Representative images and corresponding statistical plots are shown. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. SNGH20(−)-NC group, ^**P < 0.01 vs. SNHG20(−)-NC group, ^#P < 0.05 vs. SNHG20(+)-NC group, ^##P < 0.01 vs. SNHG20(+)-NC group. Scale bars indicate 50 μm. g Three-dimensional culture was used to measure the effect of VM on U87 and U251 cells by SNHG20. Representative images and corresponding statistical plots are shown. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. SNHG20(−)-NC group, ^#P < 0.05 vs. SNHG20(+)-NC group. Scale bars indicate 50 μm

Fig. 3

ZRANB2 strengthened the malignant behaviors of glioma cells by stabilizing SNHG20. a RNA-IP confirmed the binding interaction between ZRANB2 and SNGH20. Relative enrichment was measured by qRT-PCR; data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. anti-normal IgG respective group. b The graph shows nascent SNHG20 in U87 and U251 cells; data are presented as mean ± SD (n = 3, each group). c The graph shows SNHG20 levels at different times treated by ActD in U87 and U251 cells (regulated by ZRANB2); data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. ZRANB2(−)-NC group, ^##P < 0.01 vs. ZRANB2(+)-NC group. d Protein levels of MMP1, MMP9 and VE-Cadherin regulated by ZRANB2 and SNHG20 in U87 and U251 cells. Representative protein expressions and corresponding IDVs of MMP1, MMP9, VE-Cadherin in U87 and U251 are shown; data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. ZRANB2(−) + SNHG20(+) group, ^**P < 0.01 vs. ZRANB2(−) + SNHG20(+) group, ^#P < 0.05 vs. ZRANB2(+) + SNHG20(−) group, ^##P < 0.01 vs ZRANB2(+) + SNHG20(−) group. e CCK-8 assay was used to measure the effect of proliferation on U87 and U251 cells by ZRANB2 and SNHG20. Data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. ZRANB2(−) + SNHG20(+) group, ^##P < 0.01 vs. ZRANB2(+) + SNHG20(−) group. f Transwell assay was used to measure the effect of migration and invasion on U87 and U251 cells by ZRANB2 and SNHG20. Representative images and corresponding statistical plots are shown. Data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. ZRANB2(−) + SNHG20(+) group, ^##P < 0.01 vs ZRANB2(+) + SNHG20(−) group. Scale bars indicate 50 μm. g Three-dimensional culture was used to measure the effect of VM on U87 and U251 cells by ZRANB2 and SNHG20. Representative images and corresponding statistical plots are shown. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. ZRANB2(−) + SNHG20(+) group, ^#P < 0.05 vs. ZRANB2(+) + SNHG20(−) group. Scale bars indicate 50 μm

Fig. 4

Endogenous expression of FOXK1 and effect of FOXK1 on the biological behaviors of glioma cells. a Protein levels of FOXK1 in NBTs, LGGTs and HGGTs. Representative protein expressions and corresponding IDVs of FOXK1 in NBTs (n = 15), low-grade glioma (n = 15), and high-grade glioma (n = 15) are shown; data are presented as mean ± SD.^**P < 0.01 vs. NBTs group, ^##P < 0.01 vs. LGGTs group. b Protein levels of FOXK1 in NHA, U87 and U251 cells. Data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. NHA group. c Gene expression difference curve according to TCGA. d Survival analysis curve according to TCGA. e Effects of ZRANB2 on FOXK1 expression; data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. ZRANB2(−)-NC group, ^**P < 0.01 vs. ZRANB2(−)-NC group, ^##P < 0.01 vs. ZRANB2(+)-NC group. f Effects of SNHG20 on FOXK1 expression; data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. SNHG20(−)-NC group, ^##P < 0.01 vs. SNHG20(+)-NC group. g FOXK1 expression regulated by ZRANB2 and SNHG20; data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. ZRANB2(−) + SNHG20(+) group, ^#P < 0.05 vs. ZRANB2(+) + SNHG20(−) group, ^##P < 0.01 vs. ZRANB2(+) + SNHG20(−) group. h Protein levels of MMP1, MMP9 and VE-Cadherin regulated by FOXK1. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. FOXK1(−)-NC group, ^**P < 0.01 vs. FOXK1(−)-NC group, ^#P < 0.05 vs. FOXK1(+)-NC group, ^##P < 0.01 vs. FOXK1(+)-NC group. i CCK-8 assay. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. FOXK1(−)-NC group, ^**P < 0.01 vs. FOXK1(−)-NC group, ^#P < 0.05 vs. FOXK1(+)-NC group, ^##P < 0.01 vs. FOXK1(+)-NC group. j Transwell assay. Representative images and corresponding statistical plots are shown. Data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. FOXK1(−)-NC group, ^#P < 0.05 vs. FOXK1(+)-NC group, ^##P < 0.01 vs. FOXK1(+)-NC group. Scale bars indicate 50 μm. k Three-dimensional culture. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. FOXK1(−)-NC group, ^#P < 0.05 vs. FOXK1 (+)-NC group. Scale bars indicate 50 μm

Fig. 5

SNHG420 strengthened the malignant behaviors of glioma cells by degrading FOXK1 mRNA through SMD pathway. a The predicted SNHG20 binding site in FOXK1 mRNA 3’UTR and results of dual-luciferase reporter assays. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. FOXK1–3’UTR-Wt + SNHG20(+)-NC group. b RNA-IP results. Data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. anti-normal IgG group. c RNA-IP results. Data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. anti-normal IgG group. d Stability of FOXK1 mRNA by SNHG20; data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. SNHG20(−)-NC group, ^##P < 0.01 vs. SNHG20(+)-NC group. e Stability of FOXK1 mRNA by STAU1; data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. SNHG20(−)-NC group. f Stability of FOXK1 mRNA by UPF1; data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. SNHG20(−)-NC group. g Effect of STAU1 on FOXK1 expression; data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. STAU1(−)-NC group, ^**P < 0.01 vs. STAU1(−)-NC group. h Effect of UPF1 on FOXK1 expression; data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. UPF1(−)-NC group. i Protein levels of MMP1, MMP9 and VE-Cadherin. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. SNHG20(−) + FOXK1(−) group, ^**P < 0.01 vs. SNHG20(−) + FOXK1(−) group, ^#P < 0.05 vs. SNHG20(+) + FOXK1(+) group, ^##P < 0.01 vs. SNHG20(+) + FOXK1(+) group. (J) CCK-8. Data are presented as mean ± SD (n = 3, each group). ^**P < 0.01 vs. FOXK1(−)-NC group, ^#P < 0.05 vs. FOXK1(+)-NC group, ^##P < 0.01 vs. FOXK1(+)-NC group. k Transwell. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. SNHG20(−) + FOXK1(−) group, ^**P < 0.01 vs. SNHG20(−) + FOXK1(−) group, ^#P < 0.05 vs. SNHG20(+) + FOXK1(+) group, ^##P < 0.01 vs. SNHG20(+) + FOXK1(+) group. Scale bars indicate 50 μm. l Three-dimensional culture. Data are presented as mean ± SD (n = 3, each group). ^*P < 0.05 vs. SNHG20(−) + FOXK1(−) group, ^**P < 0.01 vs. SNHG20(−) + FOXK1(−) group, ^##P < 0.01 vs. SNHG20(+) + FOXK1(+) group. Scale bars indicate 50 μm

Fig. 6

FOXK1 bound to the promoters of MMP1, MMP9 and VE-Cadherin in U87 and U251 cells. a Schematic representation of the MMP1 promoter region in 3000 bp upstream of the transcription start site (TSS) designated as + 1. Putative FOXK1 binding sites are shown. Immunoprecipitated DNA was amplified by PCR using rabbit IgG as negative control. b Schematic representation of the MMP9 promoter region in 3000 bp upstream of the transcription start site (TSS) designated as + 1. Putative FOXK1 binding sites are shown. Immunoprecipitated DNA was amplified by PCR using rabbit IgG as negative control. c Schematic representation of the VE-Cadherin promoter region in 3000 bp upstream of the transcription start site (TSS) designated as + 1. Putative FOXK1 binding sites are shown. Immunoprecipitated DNA was amplified by PCR using rabbit IgG as negative control

Fig. 7

The stable expressing cells were used for tumor xenografts study in vivo. a The nude mice sample tumor from respective group was shown. b Tumor growth curves in nude mice were shown. Tumor volume was calculated every five days after injection and tumor was excised after 45 days; data are presented as mean ± SD (n = 8, each group). ^**P < 0.01 vs. ZRANB2(−)-NC + SNHG20(−)-NC group, ^##P < 0.01 vs. ZRANB2(−) group, ^&&P < 0.01 vs. SNHG20(−) group. c The survival curves of nude mice that were injected into the right striatum were shown (n = 8, each group). d CD34-PAS staining was used to detect the VM in xenografted tumor; data are presented as mean ± SD (n = 5, each group). ^*P < 0.05 vs. ZRANB2(−)-NC + SNHG20(−)-NC group, ^**P < 0.01 vs. ZRANB2(−)-NC + SNHG20(−)-NC group, ^##P < 0.01 vs. ZRANB2(−) group, ^&&P < 0.01 vs. SNHG20(−) group. Scale bars indicate 25 μm

Fig. 8

The schematic illusion of interactions between ZRANB2, SNHG20 and FOXK1 in glioma