Overexpression of TIP30 inhibits the growth and invasion of glioma cells

Abstract

Glioma is an aggressive malignancy with limited effective treatment and poor prognosis. Therefore, the identification of novel prognostic markers and effective therapeutic targets is important for the treatment of human glioma. TIP30 is a tumor suppressor involved in the regulation of numerous cellular processes, including tumor cell growth, metastasis, and angiogenesis in various human cancers. The present study investigated whether Tat‑interacting protein (TIP)30 was able to regulate tumorigenesis and predict the clinical outcome of patients with glioma. A total of 92 human glioma tissue samples and 10 normal brain tissue samples were examined by immunostaining. The results indicated that the expression levels of TIP30 significantly decreased in glioma tissue samples. as compared with normal brain tissue samples. Furthermore, TIP30 expression was inversely correlated with tumor histological classification, pathological grade, tumor size, and epidermal growth factor receptor (EGFR) expression; however, no association was detected between TIP30 expression and patient age and gender. In addition, patients with positive TIP30 expression exhibited significantly longer median overall survival rates, as compared with those with negative TIP30 expression. In vitro experiments revealed that upregulation of TIP30 expression by lentiviral vector transfection inhibited cell growth and induced cell apoptosis, as determined by MTT assay and Annexin V‑fluorescein isothiocyanate staining, respectively. In addition, TIP30 expression markedly attenuated cell migration and invasion, as determined by wound healing and transwell assays. Upregulation of TIP30 expression in glioma cells decreased the expression levels of EGFR and its associated downstream molecules phosphorylated extracellular signal‑regulated kinases (ERK) and phosphorylated AKT, as determined by western blot analysis. The results of the present study indicated that TIP30 may suppress oncogenesis and glioma progression, thereby improving the prognosis of patients with glioma. Therefore, TIP30 may prove useful as a prognostic biomarker, and as a potential target for glioma therapy.

Figure 1

Expressions of Tat-interacting protein (TIP)30 and epidermal growth factor receptor (EGFR) in glioma and normal brain tissue samples. (A) (a) Negative expression of TIP30 in glioma tissue samples. (b) Positive expression of TIP30 in normal tissue samples. (c) Strong positive expression of EGFR in glioma tissue samples. (d) Weak positive expression of epidermal growth factor receptor (EGFR) in normal brain tissue samples. (Magnification, ×400) (B) Protein expression of TIP30 and EGFR in glioma and normal brain tissue samples (P<0.05). (C) Median overall survival (mOS) of patients with TIP30 and EGFR-positive and TIP30 and EGFR-negative expression. (Left panel) Patients with TIP30-positive expression exhibited longer mOS times, as compared with patients with TIP30-negative expression (P=0.019), and (right panel) patients with EGFR-positive expression exhibited shorter mOS times, as compared with patients with EGFR-negative expression (P=0.021).

Figure 2

Overexpression of Tat-interacting protein (TIP)30 inhibits glioma cell growth. (A) mRNA expression levels of TIP30 were analyzed by real time-quantitative polymerase chain reaction in lentivirus (LV)-TIP30, mock and LV-control (Con) U87 glioma cells. (B) Protein expression levels of TIP30 were analyzed by western blotting. (C) Overexpression of TIP30 inhibits glioma cell growth. Four days following virus infection, the U87 cells were seeded in 96-well plates in order to carry out an MTT assay. ^*P<0.05 LV-TIP30 group, vs. the LV-Con group. (D) Cell apoptosis was detected by flow cytometry. The cells were assessed by flow cytometry following Annexin V staining. Q4-2, early apoptotic cells positively stained for Annexin V-fluorescein isothiocyanate (FITC), and negative for propidium iodide (PI); Q3-2, normal cells not stained by Annexin V-FITC or PI; Q2-2, necrotic cells and late apoptotic cells stained by both Annexin V-FITC and PI; (E) Data are presented as the mean ± standard deviation of three independent experiments. ^*P<0.05 and ^**P>0.05.

Figure 3

Overexpression of Tat-interacting protein (TIP)30 inhibits the migration and metastasis of U87 glioma cells. (A) U87 cells infected with lentivirus (LV)-TIP30 and LV-control (Con) were examined in a wound healing assay. Images were captured at 0, 18, and 36 h. (B) U87 cells infected with LV-TIP30 and LV-Con were incubated in Matrigel for 48 h, and the number of cells invading the membrane was calculated from five randomly selected microscopic fields. (C) The number of cells invading through the Matrigel are presented as the mean ± standard deviation. ^*P<0.05.

Figure 4

U87 glioma cell infection with lentivirus (LV)-Tat-interacting protein (TIP)30 induced significant downregulation of epidermal growth factor receptor (EGFR)/phosphorylated (p) extracellular signal-regulated kinases (ERK)/pAKT. The expression levels of EGFR, pERK, and pAKT were examined by western blotting in LV-TIP30 and LV-control (Con) U87 cells.