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Glioblastoma multiforme (GBM) is the most common and most malignant type of primary adult brain cancer. The most common phenotype associated with GBM is cellular invasion; however, the molecular mechanisms governing this process are poorly understood. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a nuclear protein with roles in cellular proliferation and mitotic spindle assembly. TPX2 is overexpressed in various malignancies, including human malignant astrocytoma. Despite this finding, the exact role of TPX2 in human glioma is not well defined. The present study reports the elevated expression of TPX2 in a number of glioma cell lines. TPX2 overexpression promoted cellular proliferation, decreased the percentage of cells in G0/G1 phase, and increased invasion of both U251 and U87 cells. Overexpression of TPX2 also significantly enhanced the phosphorylation of AKT, decreased the expression of p21, and increased the expression of cyclin D1 and matrix metallopeptidase (MMP)-9. In both U251 and U87 cells, knockdown of TPX2 resulted in phenotypes that are in direct contrast to those observed following TPX2 overexpression. Specifically, TPX2 knockdown inhibited cell proliferation, increased the percentage of cells in G0/G1 phase, inhibited invasion, decreased AKT phosphorylation, decreased the expression of MMP-9 and cyclin D1, and increased p21 expression. The AKT inhibitor IV in large part phenocopied the effect of TPX2 knockdown. The present data suggest that TPX2 promotes glioma cell proliferation and invasion via AKT signaling.
Figure 1. TPX2 expression in primary NHAs and glioma cell lines. (A) TPX2 mRNA expression in primary NHAs and in and glioma cell lines (U251, U87 and LN229) was assessed by RT-qPCR. RT-qPCR revealed that TPX2 was upregulated in all the glioma cell lines compared with the NHA cells. *P<0.05 vs. NHAs. (B) TPX2 protein expression in primary NHAs and glioma cell lines (U251, U87 and LN229) was detected by western blotting. NHA, normal human astrocyte; TPX2, targeting protein for Xenopus kinesin-like protein 2; mRNA, messenger RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Figure 2. TPX2 overexpression promotes cell proliferation in U251 and U87 cells. (A and B) TPX2 overexpression significantly promotes the proliferation of (A) U251 and (B) U87 glioma cells at different time points. (C and D) TPX2 overexpression led to a reduced number of (C) U251 and (D) U87 glioma cells in G0/G1 phase and an increased number of cells in S phase. Data are presented as the mean +standard deviation of three independent experiments. *P<0.05. TPX2, targeting protein for Xenopus kinesin-like protein 2; OD, optical density.
Figure 3. TPX2 overexpression promotes invasion in U251 and U87 cells. (A) U251 and (B) U87 cells were examined for cell invasion in 24-well plates with Transwell chambers. (C) The migrated cells were stained with crystal violet and observed under a light microscope (magnification, Ã200). The invasiveness of U251 and U87 cells was enhanced with TPX2 overexpression. *P<0.05. TPX2, targeting protein for Xenopus kinesin-like protein 2.
Figure 4. TPX2 overexpression activates AKT signaling pathway in (A) U251 and (B) U87 cells. (C) Western blot analysis of TPX2, pAKT, AKT, p21, cyclin D1 and MMP-9 expression in cells transfected with pcDNA-TPX2 or pcDNA3.1. β-actin served as the loading control (*P<0.05). TPX2, targeting protein for Xenopus kinesin-like protein 2; MMP, matrix metallopeptidase; p, phosphorylated.
Figure 5. TPX2 knockdown suppresses cell proliferation in U251 and U87 cells. (A and B) TPX2 knockdown significantly suppressed the proliferation of (A) U251 and (B) U87 glioma cells at different time points. (C and D) TPX2 knockdown resulted in an increased number of (C) U251 and (D) U87 glioma cells in G0/G1 phase and a reduced number of cells in S phase. Data are the mean + standard deviation of three independent experiments. *P<0.05. TPX2, targeting protein for Xenopus kinesin-like protein 2; Con, control; siRNA, small interfering RNA; OD, optical density.
Figure 6. TPX2 knockdown suppresses the invasion capacity of (A) U251 and (B) U87 cells. (A-C) U251 and U87 cells were examined for cell invasion in 24-well plates with Transwell chambers. (C) The migrated cells were stained with crystal violet and observed under a light microscope (magnification, Ã200). The invasiveness of U251 and U87 cells was attenuated with TPX2 knockdown. *P<0.05. TPX2, targeting protein for Xenopus kinesin-like protein 2; Con, control; siRNA, small interfering RNA.
Figure 7. TPX2 knockdown inhibits AKT signaling pathway in (A) U251 and (B) U87 cells. (C) Western blot analysis of TPX2, pAKT, AKT, p21, cyclin D1 and MMP-9 expression in cells transfected with TPX2-siRNA or Con-siRNA. β-actin served as the loading control (*P<0.05). TPX2, targeting protein for Xenopus kinesin-like protein 2; MMP, matrix metallopeptidase; p, phosphorylated; Con, control; siRNA, small interfering RNA.
Figure 8. AKT inhibitor IV inhibits TPX2 expression and AKT signaling pathway in (A) U251 and (B) U87 cells. (C) Western blot analysis of TPX2, pAKT, AKT, p21, cyclin D1 and MMP-9 expression following AKT inhibitor IV treatment. β-actin served as the loading control (*P<0.05). TPX2, targeting protein for Xenopus kinesin-like protein 2; MMP, matrix metallopeptidase; p, phosphorylated; Con, control; siRNA, small interfering RNA.
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