Depletion of thymopoietin inhibits proliferation and induces cell cycle arrest/apoptosis in glioblastoma cells

Abstract

Background: Glioblastoma (GBM) is the most malignant nervous system tumor with an almost 100 % recurrence rate. Thymopoietin (TMPO) has been demonstrated to be upregulated in various tumors, including lung cancer, breast cancer, and so on, but its role in GBM has not been reported. This study was aimed to determine the role of TMPO in GBM.

Methods: Publicly available Oncomine dataset analysis was used to explore the expression level of TMPO in GBM specimens. Then the expression of TMPO was knocked down in GBM cells using lentiviral system, and the knockdown efficacy was further validated by real-time quantitative PCR and western blot analysis. Furthermore, the effects of TMPO silencing on GBM cell proliferation and apoptosis were examined by MTT, colony formation, and flow cytometry analysis. Meanwhile, the expression of apoptotic markers caspase-3 and poly(ADP-ribose) polymerase (PARP) were investigated by western blot analysis.

Results: This study observed that the expression of TMPO in GBM specimens was remarkably higher than that in normal brain specimens. Moreover, knockdown of TMPO could significantly inhibit cell proliferation and arrest cell cycle progression at the G2/M phase. It also found that TMPO knockdown promoted cell apoptosis by upregulation of the cleavage of caspase-3 and PARP protein levels which are the markers of apoptosis.

Conclusions: The results suggested TMPO might be a novel therapeutic target for GBM.

Keywords: Apoptosis; Cell proliferation; Glioblastoma; TMPO.

Fig. 1

Upregulation of TMPO mRNA in GBM was revealed by Oncomine database. The expression of the TMPO gene was extracted and analyzed from a the French Brain dataset, b the Liang Brain dataset, c the Murat Brain dataset, d the TCGA Brain dataset, e the Shai Brain dataset, f the Sun Brain dataset, and g the Pomeroy Brain dataset shown as scatterplots

Fig. 2

Knockdown efficiency of TMPO by lentivirus infection in the GBM cells. a Quantitative real-time PCR analysis of TMPO knockdown efficiency in U251 and U87 cells. The mRNA expression of TMPO was significantly suppressed when the cells were infected with TMPO-shRNA. b Western blot analysis validated TMPO-shRNA effectively downregulated the protein level of TMPO in U251 and U87 cells. c Western blot analysis confirmed TMPO-shRNA(S2) effectively decreased the protein expression of TMPO in U251 cells. **p < 0.01; scale bars, 10 μm

Fig. 3

Downregulation of TMPO inhibited the proliferation of GBM cells. a, b Cell proliferation in U251 and U87 cells after TMPO-shRNA infection was determined by MTT assay, respectively. c The proliferation of U251 cells was examined after TMPO-shRNA(S2) infection by MTT assay. d Representative microscopic images of colonies in TMPO-shRNA- or TMPO-shRNA(S2)-infected U251 cells were stained by crystal violet. e Statistical analysis of the number of colonies. **p < 0.01, ***p < 0.001; scale bars, 25 μm

Fig. 4

Downregulation of TMPO arrested the cell cycle of U251 cells. a, d The cell cycle distribution determined by flow cytometer. b, e Statistical analysis of the cell cycle in the U251 cells with TMPO knockdown. c, f The respective proportion of sub-G1 phase cells. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Downregulation of TMPO induced the cell apoptosis of U251 cells. a, b Representative images showing Annexin V/7-ADD staining results in TMPO-shRNA- or TMPO-shRNA(S2)-infected U251 cells. c, d Statistical analysis of apoptosis in the U251 cells with TMPO knockdown. e The protein expression of cleaved PARP and cleaved caspase-3 in U251 cells was significantly increased after knockdown of the TMPO gene as verified by western blot. **p < 0.01, ***p < 0.001