miR-504 suppresses mesenchymal phenotype of glioblastoma by directly targeting the FZD7-mediated Wnt-β-catenin pathway

Abstract

Background: MicroRNAs (miRNAs) play crucial roles in tumor initiation and development. Previously, we indicated that miR-504 is downregulated and suppresses tumor proliferation in glioblastoma (GBM). However, the regulation and relevant mechanism of miR-504 in GBM mesenchymal (ME) transition remain unclear.

Methods: Transcriptome and clinical data were obtained from The Cancer Genome Atlas (TCGA) database. The potential functions of miR-504 were predicted using gene ontology analysis. GBM cell migration and invasion were examined using wound healing and Transwell assays. Epithelial-mesenchymal transition (EMT) progression in GBM cell lines was detected with immunofluorescence and western blotting. The stemness activity of glioma stem-like cells (GSCs) was assessed by sphere formation assay and tumor xenograft model. miR-504 binding to the FZD7 (frizzled class receptor 7) 3' untranslated region (3'UTR) was validated using dual luciferase reporter assay. TOP/FOP Flash assays were conducted to determine the effects of miR-504 on Wnt/β-catenin signaling.

Results: Analysis of TCGA transcriptomic data showed that low miR-504 expression correlated with ME subtype transition and poor survival in patients with GBM. Functional experiments showed that miR-504 overexpression suppressed malignant behaviors of GBM cells, such as migration, invasion, EMT, and stemness activity. Furthermore, miR-504 was a negative regulator of the Wnt-β-catenin pathway by directly repressing FZD7 expression, and FZD7 overexpression reversed the EMT inhibition caused by miR-504. Moreover, the low miR-504/FZD7 expression ratio was a ME subtype marker and could serve as a significant prognostic indicator and predict the clinical outcome of chemotherapy and radiotherapy for patients with GBM in TCGA dataset.

Conclusions: Our results suggest that miR-504 suppresses the aggressive biological processes associated with the ME phenotype of GBM and could be a potential candidate for therapeutic applications in these malignant brain tumors.

Keywords: EMT; FZD7; GBM; Mesenchymal phenotype; Wnt–β-catenin pathway; miR-504.

Fig. 1

Downregulation of miR-504 in ME subtype GBM. a Analysis of TCGA data of miR-504 expression levels in NBT (n = 10) and GBM (n = 517). b Analysis of TCGA data of miR-504 expression levels in ME subtype GBM (n = 172) and PN subtype GBM (n = 105). c PCA based on the miR-504–related genes shows a distinct distribution pattern between patients with the ME and the PN subtype. d The expression pattern of ME and PN signature genes corresponding to miR-504 expression. e Pearson correlation analysis between miR-504 and ME score in TCGA. f Kaplan–Meier analysis of overall survival of patients with GBM from TCGA database according to miR-504 expression level. g GO analysis of the top 200 differentially upregulated genes in miR-504 low-expression tumors. h GO analysis of top 200 genes negatively related to miR-504. i–l GSEA highlighting positive association of decreased miR-504 expression levels with EMT, cell adhesion, and angiogenesis. ***P < 0.001

Fig. 2

miR-504 suppresses the ME properties and EMT of GBM cells. a qRT-PCR detection of miR-504 expression in U87 and U373 cells transfected with miR-504 overexpression and inhibition vectors. a Representative images of GBM cell morphological changes after miR-504 transfection (scale bars = 100 μm). (c, d) Representative results of wound healing assays (c) and Transwell assays (d) showing the effect of miR-504 overexpression and inhibition on the migration and invasive abilities of GBM cells (scale bars = 100 μm). e Western blot analysis of the levels of representative epithelial and ME markers. f Immunofluorescence examination of ME marker expression in U87 and U373 cells transfected with miR-504 or miR-NC (red, vimentin; blue, DAPI; scale bars = 100 μm). Data are the mean ± SD of three independent experiments. *P < 0.05,**P < 0.01, ***P < 0.001

Fig. 3

miR-504 attenuates GSC stemness activity. a Immunofluorescence showing tumor spheres comprised of CD133-positive GSCs (red, CD133; blue, DAPI; scale bars = 100 μm). b GSCs differentiated into GFAP-positive cells after serum induction (red, GFAP; blue, DAPI; scale bars = 100 μm). c qRT-PCR detection of miR-504 expression in GSC-U87 and GSC-1295 cells transfected with miR-504 overexpression and inhibition vectors. d Representative images of tumor spheres formed after 14-days culture (scale bars = 500 μm). e Western blot analysis of the levels of the representative stem cell markers. f–h miR-504 reduced GSC-U87 cell tumorigenicity in vivo. i Immunohistochemistry for CD133 and KLF4 in xenograft sections (scale bar = 50 μm). Data are the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

miR-504 targets the FZD7 3′UTR directly. a Venn diagram identifying the overlap between predicted genes and miR-504–negatively related genes. b The predicted target site of FZD7 3′UTR binding to miR-504. c Pearson correlation analysis of miR-504 and FZD7 in TCGA. d TCGA data analysis of FZD7 expression levels between miR-504 high-expression tumors (n = 129) and miR-504 low-expression tumors (n = 388). e Measurement of firefly/Renilla luciferase activity in HEK-293 T cells co-transfected with wild-type-FZD7–3′-UTR or mutant-type-FZD7–3′-UTR reporters and NC mimic or miR-504 mimic. f, g qRT-PCR determination of FZD7 mRNA levels in U87 and U373 cells after transfection with miR-504, miR-NC, anti-NC, or anti-miR-504. h Western blot analysis of FZD7 protein levels in U87 and U373 cells after transfection with miR-504, miR-NC, anti-NC or anti-miR-504 . i Immunofluorescence examination of FZD7 expression in U87 cells transfected with miR-504 or miR-NC (red, FZD7; green, GFP; blue, DAPI; scale bars = 100 μm). j Immunohistochemistry of FZD7 in xenograft sections (scale bar = 50 μm). Data are the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

miR-504 regulates Wnt–β-catenin signaling activation. a TOP/FOPFlash reporter activity assay detection of relative Wnt signaling activity in U87 and U373 cells. b Western blot assessment of the effects of miR-504 on β-catenin, p-GSK3β, GSK3β, and c-MYC protein levels in U87 and U373 cells. c, d Western blot (c) and immunofluorescence (d) assay detection of nuclear accumulation of β-catenin in U87 cells. e, f qRT-PCR analysis of the relative mRNA expression of downstream target genes of the Wnt–β-catenin pathway, i.e., CD44 (e) and c-MYC (f), in U87 and U373 cells. Data are the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

miR-504 inhibits GBM cell ME transition by suppressing the FZD7-mediated Wnt–β-catenin pathway. a, b qRT-PCR (a) and western blot (b) of FZD7 expression in miR-504–transfected U87 and U373 cells after FZD7 transfection. c, d Wound healing assay (c) and Transwell assay (d) evaluation of the restorative effect of FZD7 on miR-504–induced inhibition of migration and invasion in U87 and U373 cells (scale bar = 100 μm). e Western blot detection of EMT markers in U87 and U373 cells co-transfected with miR-504 and FZD7. f TOP/FOPFlash reporter activity detection of relative Wnt signaling activity in U87 and U373 cells. g Western blot detection of β-catenin, p-GSK3β, and GSK3β expression levels in miR-504 and FZD7 co-transfected cells. h Western blot assessment of the effects of FZD7 on nuclear β-catenin, c-MYC, and CD44 protein levels in U87 cells. i The diagram illustrated that miR-504 decreased the EMT and stemness via inhibiting the FZD7 mediated Wnt/β-catenin signaling pathway activity. Data are the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

Identification of the miR-504/FZD7 ratio as a ME subtype marker in GBM. a Distribution of the miR-504/FZD7 ratio between ME subtype GBM (n = 172) and PN subtype GBM (n = 105) in TCGA. b Chi-square analysis of the relationship between the miR-504/FZD7 ratio and molecular subtype. c The expression patterns of ME and PN signature genes corresponding to the miR-504/FZD7 ratio. d Pearson correlation analysis of the miR-504/FZD7 ratio and ME score in TCGA. e PCA based on ME signature genes and EMT signature genes showed distinct distribution patterns between GBM of high- and low-ratio groups. f GO analysis of top 200 genes negatively related to the miR-504/FZD7 ratio. g GO analysis of top 200 differentially upregulated genes in low–miR-504/FZD7 ratio tumors in TCGA. h GSEA comparison of ME phenotype between high- and low–miR-504/FZD7 ratio cases. i Clustering of GSVA scores and analyses of biological processes associated with the miR-504/FZD7 ratio in GBM according to TCGA dataset (GO:0007158, neuron cell–cell adhesion; GO:2000047, regulation of cell–cell adhesion mediated by cadherin; GO:0060231, mesenchymal to epithelial transition; GO:0097150, neuronal stem cell population maintenance; GO:2000036, regulation of stem cell population maintenance). ***P < 0.001

Fig. 8

The miR-504/FZD7 ratio is a prognostic indicator in GBM. a Kaplan–Meier assessment of the association between the miR-504/FZD7 ratio and overall survival of patients with GBM in TCGA. b–d Prognostic significance of the miR-504/FZD7 ratio in cohorts stratified by age (b), sex (c), and MGMT promoter status (d). e Kaplan–Meier analysis of survival of chemotherapy-treated patients with GBM based on the miR-504/FZD7 ratio. f Comparison of 1–5-year survival rates between high and low miR-504/FZD7 ratio groups in chemotherapy-treated patients with GBM. g Kaplan–Meier analysis of overall survival according to the miR-504/FZD7 ratio of standard radiotherapy–treated patients with GBM from TCGA. h Comparison of 1–5-year survival rates between high and low miR-504/FZD7 ratio groups in standard radiotherapy–treated patients with GBM. *P < 0.05, **P < 0.01, ***P < 0.001