β-transducin repeat-containing E3 ubiquitin protein ligase inhibits migration, invasion and proliferation of glioma cells

Abstract

β-transducin repeat-containing E3 ubiquitin protein ligase (β-TrCP) serves as the substrate recognition subunit for the Skp1-Cullin1-F-box protein E3 ubiquitin ligase, which recognizes the double phosphorylated DSG (X)_2+nS destruction motif in various substrates that are essential for numerous aspects of tumorigenesis and regulates several important signaling pathways. However, the biological significance of β-TrCP in glioma progression remains largely unknown. A previous study by the authors demonstrated that the levels of β-TrCP protein expression in brain glioma tissues were significantly lower compared with non-tumorous tissues and that higher grades of gliomas exhibited lower levels of β-TrCP expression in comparison with lower glioma grades. In addition, low β-TrCP expression was associated with poor prognosis in patients with glioma. Subsequently, the present study aimed to investigate the effect of β-TrCP on migratory, invasive and proliferative abilities of glioma cells. β-TrCP plasmids were transfected into cultured U251 and U87 glioma cells, and changes in migration, invasion and proliferation were analyzed using wound healing, Transwell and EdU assays. It was identified that the overexpression of β-TrCP inhibited migration, invasion and proliferation in glioma cells. In summary, these results indicate that β-TrCP may serve a protective role against the progression of glioma by suppressing cell migration, invasion and proliferation. The potential mechanism of β-TrCP I glioma cells requires additional investigation.

Keywords: glioma cells; invasion; migration; proliferation; β-transducin repeat-containing E3 ubiquitin protein ligase.

Figure 1.

Western blot analysis of exogenous β-TrCP expression in U251 and U87 cells. After 24 h transfection with β-TrCP plasmids, the cells were lysed and protein extraction was performed. Western blot analysis was carried out using (A) Flag or (B) β-TrCP antibody. β-actin was used as the loading control. β-TrCP, β-transducin repeat-containing protein; exo, exogenous, endo, endogenous.

Figure 2.

Overexpression of β-TrCP significantly inhibits the migratory and invasive ability of glioma cells. (A) Wound healing assay of U251 and U87 cells at 0 and 24 h following transient transfection with β-TrCP plasmids vs. the control vector group. Scale bar, 100 µm. (B) Cell migration was quantified at 24 h. (C) Representative micrographs of U251 and U87 invasion at 24 h after transient transfection with β-TrCP plasmids. Scale bar, 100 µm. (D) Cell invasion was calculated from Transwell assays with Matrigel. Cell count was normalized to the vacant vector group. *P<0.05, **P<0.01 vs. control. β-TrCP, β-transducin repeat-containing protein. Scale bar, 100 µm.

Figure 3.

Overexpression of β-TrCP significantly inhibits the proliferative ability of glioma cells. Representative images of EdU positive cells: (A) U251 and (C) U87 cells. Graph presenting the changes between cells overexpressing β-TrCP and the control: (B) U251 and (D) U87 cells. *P<0.05, **P<0.01 vs. control. β-TrCP, β-transducin repeat-containing protein. Scale bar, 100 µm.