VAV3 regulates glioblastoma cell proliferation, migration, invasion and cancer stem‑like cell self‑renewal

Abstract

Glioblastoma multiforme (GBM; World Health Organization grade IV) is one of the most common and aggressive malignant brain tumors and has no effective treatment. Therefore, elucidation of the molecular mechanism of glioma development is very important for finding new therapeutic strategies. The present study evaluated the expression level of Vav guanine nucleotide exchange factor 3 (VAV3) using bioinformatics analysis and demonstrated that VAV3 was overexpressed in human glioblastoma and associated with patient survival. Knock down of VAV3 using shRNA in glioblastoma cells significantly inhibited glioblastoma cell migration, invasion and proliferation. Furthermore, downregulation of VAV3 expression inhibited the stem cell self‑renewal capacity and decreased the expression levels of the stem cell markers Nestin and Sox2. Bioinformatic analysis demonstrated that VAV3 was a target gene of miR‑218. Furthermore, overexpression of VAV3 markedly reversed the tumor suppressor effect of miR‑218 in glioblastoma cell. These findings suggested that VAV3 could be a potential biomarker and therapeutic target for glioblastoma.

Keywords: Vav guanine nucleotide exchange factor 3; glioblastoma multiforme; miR‑218; proliferation; stemness.

Figure 1.

VAV3 expression in human glioblastoma. (A) VAV3 mRNA expression levels in human glioblastoma and normal tissues from TCGA database. (B) VAV3 mRNA expression levels in human glioblastoma in the CGGA database. Kaplan-Meier survival analysis demonstrated that VAV3 expression was negatively correlated with patient survival in data from the (C) TCGA and (D) CGGA databases. *P<0.05 and **P<0.01. VAV3, Vav guanine nucleotide exchange factor 3; GBM, glioblastoma multiforme; TCGA, The Cancer Genome Atlas; CGGA, Chinese Glioma Genome Atlas; WHO, World Health Organization.

Figure 2.

Reduced VAV3 inhibits U251 glioblastoma cell proliferation in vitro. (A) Reverse transcription-quantitative PCR analysis demonstrated the VAV3 mRNA expression level in U251 cells with endogenous VAV3 suppressed by shRNAs. (B) Western blotting analysis of VAV3 protein expression levels in U251 cells with endogenous VAV3 suppressed with shRNAs. (C) Knockdown of VAV3 inhibited cell proliferation in U251 cells. The wound healing assay demonstrated different cell migration rates in control shRNA-U251, VAV3 shRNA-1#-U251 and VAV3 shRNA-2#-U251 cells. (D) Representative images were taken at different time points. (E) Quantification of cell motility was assessed by measuring the wound width. Transwell assays of U251 cells transfected with control shRNA, VAV3 shRNA-1# and VAV3 shRNA-2#. (F) Representative fields of view of invasive cells and migratory cells. (G) Quantitative analysis of the invasive and migratory cells from three independent experiments. Scale bar=50 µm. Error bars represented standard error. ***P<0.001. VAV3, Vav guanine nucleotide exchange factor 3; shRNA, short hairpin RNA.

Figure 3.

Decreased VAV3 inhibited U87 glioblastoma cell proliferation in vitro. (A) Reverse transcription-quantitative PCR analysis demonstrated that VAV3 expression in U87 cells with endogenous VAV3 was suppressed by shRNAs. (B) Western blotting analysis of VAV3 protein expression levels in U87 cells with endogenous VAV3 suppressed by shRNAs. (C) Knockdown of VAV3 inhibited cell proliferation in U87 cells. (D) Representative fields of view of invasive cells and migratory cells. (E) Quantitative analysis of the invasive and migratory cells from three independent experiments. Scale bar=50 µm. Error bars represented standard error. ***P<0.001. VAV3, Vav guanine nucleotide exchange factor 3; shRNA, short hairpin RNA.

Figure 4.

Targeting VAV3 with shRNA decreased glioblastoma stem-like cells self-renewal. (A) The morphology of tumor spheres formed by the cancer stem cells from control shRNA U251 cells, VAV3 shRNA-1#-U251 cells and VAV3 shRNA-2#-U251 cells. (B) Glioblastoma tumor sphere diameters decreased in VAV3-shRNAs U251 GBM stemlike cells. (C) In vitro extreme limiting dilution assays to single cells demonstrated that knockdown of VAV3 in U251 cells decreased the frequency of tumor sphere formation (D) Western blotting analysis demonstrated the protein expression levels of Nestin and Sox2 in neurospheres derived from control shRNA U251, VAV3 shRNA-1#-U251 and VAV3 shRNA-2#-U251 cells. Scale bar=100 µm. Error bars represented standard error. **P<0.05. ***P<0.001. VAV3, Vav guanine nucleotide exchange factor 3; shRNA, short hairpin RNA.

Figure 5.

VAV3 shRNAs significantly decreased tumor growth in a xenograft model. (A) Photographs of tumorigenesis in U251 cells in xenograft mice. (B) Photographs of the isolated tumor tissues. (C) The volume of the xenograft tumors were measured at the indicated time points. Data are expressed as mean ± standard deviation (n=6). ***P<0.001. VAV3, Vav guanine nucleotide exchange factor 3; shRNA, short hairpin RNA.

Figure 6.

miR-218 targets VAV3 by binding to its 3′UTR. (A) Predicted miR-218 target sequences in the 3′UTR of VAV3 and mut containing eight mutated nucleotides in the 3′UTR of VAV3. (B) Reverse transcription-quantitative PCR analysis of VAV3 mRNA expression levels in glioblastoma cells transfected with miR-218 or negative control. (C) U251 cells were co-transfected with miR-218 and luciferase reporters containing either the predicted miRNA target site in VAV3 3′UTR or its corresponding MUT form. (D) Quantitative analysis of the invasive and migratory cells from three independent experiments. (E) Extreme limiting dilution assays to single cells demonstrated that VAV3 overexpression in U251MG cells increased the frequency of tumor sphere formation. Error bars represented standard error. **P<0.05. ***P<0.001. VAV3, Vav guanine nucleotide exchange factor 3; miR, microRNA; WT, wild-type; mut, mutant; con, control; NC, negative control.