GALE Promotes the Proliferation and Migration of Glioblastoma Cells and Is Regulated by miR-let-7i-5p

Abstract

Purpose: Glioma is the most common and lethal type of brain tumor. While GALE (UDP-galactose-4-epimerase) has been shown to be overexpressed in some kinds of cancers, its expression in gliomas has not been reported. MicroRNAs (miRNAs) function as tumor suppressors in many cancers, and online datasets can be used to predict whether GALE is regulated by miR-let-7i-5p. In this investigation, we explored the effect and regulatory mechanisms of GALE on glioblastoma growth via miR-let-7i-5p.

Methods: We used a Cox proportional hazards model and publicly available datasets to examine the relationship between GALE and the survival rates of glioma patients. Bioinformatics predicted the targeting of GALE by miR-let-7i-5p. The proliferation, migration, cell cycle and apoptosis of human glioblastoma cells were assessed by relevant assays. We also demonstrated the effect of GALE on glioblastoma multiforme [GBM] tumor growth using an in vivo orthotopic xenograft model.

Results: GALE was upregulated in human gliomas, especially in high-grade gliomas (e.g., GBM). An obvious decline in GALE expression was observed in human glioblastoma cell lines (U87 and U251) following treatment with a small interfering RNA (siRNA) targeting GALE or miR-let-7i-5p mimics. Knockdown of GALE or overexpression of miR-let-7i-5p (with miR-let-7i-5p mimics) inhibited U87 and U251 cell growth. miR-let-7i-5p significantly restrained the migration ability of human glioblastoma cells in vascular mimic (VM), wound healing and transwell assays, and GALE promoted glioblastoma growth in vivo.

Conclusion: Our findings confirm that GALE plays an important role in promoting the development of human glioma and that GALE can be regulated by miR-let-7i-5p to inhibit human glioblastoma growth.

Implications for cancer survivors: Our data show that cancer survivors have low GALE expression, which indicates that GALE may be a diagnostic biomarker and a promising therapeutic target in glioblastoma.

Keywords: GALE; glioblastoma; miR-let-7i-5p; migration; proliferation.

Figure 1

GALE expression in glioma is associated with tumor grade. (A) Quantitative analysis of GALE gene expression in the TCGA dataset. (B, C) The prognostic significance of GALE expression in LGG and GBM patients in the TCGA (n=667) database was analyzed. (D) IHC staining of GALE expression in gliomas of different grades and normal brain specimens. Magnification: ×200, upper; ×400, lower. *P < 0.05.

Figure 2

Silencing GALE reduces the proliferation of glioma cells. (A, B) Quantitative real-time PCR analysis showed that the relative mRNA level of GALE after si-GALE transfection was significantly decreased. GAPDH was used as the control. Western blot analysis of U87 and U251 cell lysates transfected with NC and GALE siRNAs and incubated with the GALE antibody. β-Actin was used as the control. (C, D) Growth curves of U87 and U251 cells based on the absorbance at OD₄₅₀. (E, F) The transfection was followed by EdU analysis 48 h later. (G) The clonal formation of glioblastoma cells was decreased after si-GALE therapy. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Silencing GALE reduces the migration of glioma cells. (A) A 24-h VM formation assay was performed in U87 cells. (B, E) U87 cells were transfected with NC or si-GALE. Cell migration was assessed 6 h after culture by transwell analysis. (C, D, F) U87 and U251 cells were transfected with NC or si-GALE and analyzed for wound healing during the 24-h recovery period. *P < 0.05.

Figure 4

Knockdown of GALE induces cell cycle arrest and apoptosis in human glioma cells. (A–C) U87 and U251 cells were transfected with NC or si-GALE and then subjected to cell cycle analysis by flow cytometry. Three independent experiments were carried out in each group. (D, E) Flow cytometry was used to detect the apoptosis rate of cells using the membrane protein V-FITC antibody and PI staining. (F–I) Western blot analysis of the expression levels of known cell cycle regulators, cell apoptosis markers, epithelial–mesenchymal transition (EMT) markers and possible cell signaling pathway proteins. *P < 0.05; ***P < 0.001.

Figure 5

Mir-let-7i-5p directly targets GALE. Sequence of the miR-let-7i-5p binding site and luciferase analysis of the GALE 3′-UTR. Bioinformatics was used to predict the miR-let-7i-5p binding site in the 3ʹ-UTR of GALE. (A) A 3ʹ-UTR vector was cotransfected with miR-let-7i-5p or NC miRNA into U251 cells. Relative luciferase activity was measured in the cytolytic solution 48 h after transfection. Luciferase activity levels were compared with those of NC miRNA-transfected cells, which were normalized to 1. (B–E) Quantitative real-time PCR analysis showed that the relative mRNA level of GALE after simulated transfection with miR-let-7i-5p was significantly decreased. GAPDH was used as the control. After miR-let-7i-5p mimics were transfected, GALE expression was confirmed by Western blot analysis. β-Actin was used as the control. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

Overexpression of miR-let-7i-5p reduces the proliferation of glioma cells. (A, B) EdU assays performed 48 h after transfection. (C) The clonogenicity of glioblastoma cells treated with miR-let-7i-5p mimics was decreased. (D) The expression levels of EMT markers, cell cycle regulatory factors, and other possible cell signaling pathway proteins were compared between the NC and miR-let-7i-5p mimic groups by Western blot analysis. *P < 0.05.

Figure 7

Overexpression of miR-let-7i-5p reduces the migration of glioma cells. (A) A VM formation assay of U87 cells was performed for 24 h. (B, E) U87 cells were transfected with the NC or miR-let-7i-5p mimics. Cell migration was assessed 6 h after culture by transwell analysis. (C, D, F) U87 and U251 cells were transfected with the NC or miR-let-7i-5p mimics, and wound healing assays were performed during the 24-h recovery period. *P < 0.05; ***P < 0.001.

Figure 8

Silencing GALE inhibits tumorigenesis in vivo. (A) A BALB/c nude mouse model of orthotopic xenograft glioma tumor development in each group was evaluated by a bioluminescence imaging (BLI) system. (B) The tumor size (mm³) was measured. (C) Brain sections from mouse xenografts comprised of 3 × 10⁵ U87 NC or sh-GALE cells were stained with H&E. (D) Survival rate analysis of animals implanted with cells expressing U87 NC or sh-GALE (log-rank test P<0.01; 5 animals in each group). (E–I) IHC analysis of GALE, BCL-2, Ki-67 and MMP2 in sections from the indicated xenografts. Magnification: ×200 and ×400. **P < 0.01; ***P < 0.001.