E2F transcription factor 1 elevates cyclin D1 expression by suppressing transcription of microRNA-107 to augment progression of glioma

Abstract

Background: Dysregulation of microRNAs has been frequently implicated in the progression of human diseases, including glioma. This study aims to explore the interaction between E2F transcription factor 1 (E2F1) and miR-107 in the progression of glioma.

Methods: Expression of miR-107 in glioma tissues and cells was examined. Putative binding sites between E2F1 and the promoter region of miR-107, and between miR-107 and cyclin D1 (CCND1) mRNA were predicted via bioinformatic systems and validated via chromatin immunoprecipitation and luciferase reporter gene assays. Altered expression of miR-107, E2F1, and CCND1 was introduced in A172 and T98G cells to examine their roles in cell growth and the activity of the Wnt/β-catenin signaling. In vivo experiments were performed by injecting cells in nude mice.

Results: miR-107 was poorly expressed, whereas E2F1 and CCND1 were highly expressed in glioma tissues and cells. E2F1 bound to the promoter region of miR-107 to induce transcriptional repression, and miR-107 directly bound to CCND1 mRNA to reduce its expression. Overexpression of miR-107 reduced proliferation, migration and invasion, and augmented apoptosis of glioma cells, and it reduced activity of the Wnt/β-catenin pathway. The anti-tumorigenic roles of miR-107 were blocked by E2F1 or CCND1 overexpression. Similar results were reproduced in vivo where miR-107 overexpression or E2F1 inhibition blocked tumor growth in nude mice.

Conclusion: This study suggested that E2F1 reduces miR-107 transcription to induce CCND1 upregulation, which leads to progression of glioma via Wnt/β-catenin signaling activation.

Keywords: E2F transcription factor 1; Wnt/β-catenin; cyclin D1; glioma; miR-107.

FIGURE 1

Overexpression of miR‐107 inhibits proliferation, migration, and invasion and promotes apoptosis of glioma cells. (a) Expression of miR‐107 in normal (n = 10) and glioma tissues (n = 23) examined by RT‐qPCR; (b) Expression of miR‐107 in normal brain glial cells (Heb) and in glioma cell lines (U251, A172, and T98G) examined by RT‐qPCR; (c) Expression of miR‐107 in A172 and T98G cells after miR‐107 mimic transfection examined by RT‐qPCR; (d) Proliferation of A172 and T98G cells determined by the EdU labeling assay; (e) Migration and (f) invasion abilities of A172 and T98G cells examined by the transwell assays; (g) Apoptosis rate of A172 and T98G cells determined by flow cytometry. Data were collected from three independent experiments and presented as mean ± SD. In (a), each spot indicates a sample; differences were compared by unpaired t test (a, c, d, e, f, and g) or one‐way ANOVA (b), *p < .05 versus Normal/Heb/NC mimic

FIGURE 2

E2F1 suppresses miR‐107 transcription. (a) Potential binding sites between E2F1 and the promoter region of miR‐107 (−16 to −23 bp and −19 to −26 bp) predicted on the ALGEEN system; (b) mRNA and protein levels of E2F1 in glioma tumor tissues and in normal tissues determined by RT‐qPCR and western blot analysis, respectively; (c) mRNA and protein levels of E2F1 in normal brain glial cells (Heb) and in glioma cell lines (U251, A172 and T98G) examined by RT‐qPCR and western blot analysis, respectively; (d) Expression of E2F1 mRNA and miR‐107 in A172 and T98G cells after E2F1 overexpression examined by RT‐qPCR; (e) enrichment of E2F1 at the −16 to −26 bp site at the promoter region examined by a ChIP‐qPCR assay. Data were collected from three independent experiments and presented as mean ± SD. Differences were compared by unpaired t test (b), one‐way ANOVA (c), or two‐way ANOVA (d,e), *p < .05 versus Normal/Heb/oe‐NC

FIGURE 3

miR‐107 targets CCND1 mRNA. (a) Binding sequence between miR‐107 and CCND1 mRNA predicted on the StarBase system; (b) mRNA and (c) protein levels of CCND1 in normal brain tissues (n = 10) and glioma tissues (n = 23) examined by RT‐qPCR and IHC staining, respectively; (d) mRNA and protein levels of CCND1 in normal brain glial cells (Heb) and in glioma cell lines (U251, A172, and T98G) examined by RT‐qPCR and western blot analysis, respectively; (e) Binding relationship between miR‐107 and CCND1 mRNA validated through a luciferase assay; (f) mRNA expression of CCND1 in A172 and T98G cells after oe‐E2F1 or miR‐107 mimic transfection examined by RT‐qPCR. Data were collected from three independent experiments and presented as mean ± SD. Differences were compared by unpaired t test (b and c), one‐way ANOVA (d and f), or two‐way ANOVA (e), *p < .05 versus Normal/Heb/oe‐NC; #p < .05 versus NC mimic

FIGURE 4

E2F1 regulates the miR‐107/CCND1 axis to promote malignant behaviors of glioma cells. Expression of miR‐107, E2F1 and CCND1 mRNA, and E2F1 and CCND1 protein in (a) A172 and (b) T98G cells determined by RT‐qPCR and western blot analysis, respectively; (c) Proliferation of A172 and T98G cells determined by the EdU labeling assay; (d) Migration and (e) invasion abilities of A172 and T98G cells examined by the transwell assays; (f) Apoptosis rate of A172 and T98G cells determined by flow cytometry. Data were collected from three independent experiments and presented as mean ± SD. Differences were compared by one‐way (c–f) or two‐way ANOVA (a,b), *p < .05 versus miR‐107 mimic +oe‐NC

FIGURE 5

E2F1 mediates the Wnt/β‐catenin signaling pathway. (a) Nuclear translocation of β‐catenin in A172 and T98G cells examined by immunofluorescence staining; (b) TOP/FOP activity in A172 and T98G cells determined by the TOP/FOP flash assay; (c) Protein levels of Wnt10B and β‐catenin in A172 and T98G cells detected by western blot analysis. Data were collected from three independent experiments and presented as mean ± SD. Differences were compared by one‐way ANOVA (b) or two‐way ANOVA (c), *p < .05 versus miR‐107 mimic +oe‐NC

FIGURE 6

The functions of E2F1, miR‐107 and CCND1 in tumorigenesis of glioma cells in vivo. (a) Images of the tumors in each group; (b) Volume and (c) weight of the xenograft tumors on the 21st day after cell injection; (d) Expression of miR‐107 and E2F1 and CCND1 mRNA in tumor tissues examined by RT‐qPCR; (e) Protein levels of E2F1, CCND1, Wnt10B, and β‐catenin in tumor tissues examined by western blot analysis. Data were collected from three independent experiments and presented as mean ± SD. In each group, n = 5. Differences were compared by one‐way ANOVA (b,c) or two‐way ANOVA (d,e), *p < .05 versus NC mimic; #p < .05 versus sh‐NC + oe‐NC; and p < .05 versus sh‐E2F1 + oe‐NC

FIGURE 7

A graphic abstract. E2F1 binds to the promoter region of miR‐107 to suppress its transcription, which restores the expression of the miR‐107 target gene CCND1, therefore promoting growth and metastasis of glioma cells