Study on the role of transcription factor SPI1 in the development of glioma

Abstract

Background: Glioma is a common malignant brain tumor. The purpose of this study was to investigate the role of the transcription factor SPI1 in glioma.

Methods: SPI1 expression in glioma was identified using qRT-PCR and Western blotting. Cell proliferation was assessed using the CCK8 assay. Transwell and wound healing assays were utilized to evaluate cell migration. Additionally, cell cycle and apoptosis were detected using flow cytometry.

Results: We observed that the expression level of SPI1 was up-regulated in glioma tissues, compared to normal tissues. Furthermore, we found that SPI1 is able to promote proliferation and migration of glioma cells in vitro. Flow cytometry results demonstrate that, compared to si-NC cells, si-SPI1 cells stagnated in the G1 phase, and down-regulation of SPI1 expression is able to increase rates of apoptosis. Double luciferase activity and chromatin immunoprecipitation assay results indicated that SPI1 can bind to the promoter sites and promote the proliferation and migration of glioma cells by regulating the expression of oncogenic PAICS.

Conclusions: Our results suggest that SPI1 can promote proliferation and migration of glioma. Furthermore, SPI1 can be utilized as a potential diagnostic marker and therapeutic target for glioma.

Keywords: Glioma; PAICS; SPI1; migration; proliferation.

Fig. 1.

Expression levels of SPI1 in glioblastoma tissues. a RT-qPCR analysis of SPI1 expression in glioblastoma tissues compared with the control brain tissues. Error bars indicate the mean ± standard deviation of three independent experiments. (N: normal brain tissue, T: glioma tissue, green line represents T/N=1). b The value of ΔCt was used to show the expression level of SPI1 (ΔCt =Ct (SPI1) − Ct (GAPDH)) in the 10 paired human glioblastoma tissues and control brain tissues (P < 0.01). c Western blot analysis of SPI1 protein expression in three pair glioblastoma tissues compared with the normal tissues. The differences between tumor group and normal group were tested by using independent-samplest-test. d Protein expression level of SPI1 was normalized to GAPDH and quantified using Image J. Error bars represent the mean ± SD of 3 different tests. ***, p < 0.001. e Expression levels of SPI1 mRNA with glioma tissues and control tissues in GEPIA database . *, p < 0.05. f Expression levels of SPI1 protein with glioma tissues and control tissues in UALCAN database. g Survival curve of low SPI1 expression group and high SPI1 expression group

Fig. 2.

Inhibition of SPI1 in glioma cells weakened the cell growth and metastasis in vitro. a RT-qPCR analysis of SPI1 mRNA levels after the transfection of the si-NC or si-SPI1 small interfering RNAs. SPI1 expression was normalized to GAPDH, Error bars indicate mean ± SD of 3 independent experiments.**, p< 0.01; ***, p< 0.001. b Western blot analysis of SPI1 protein levels after the transfection of the si-NC or si-SPI1 small interfering RNAs. SPI1 expression was normalized to β-Actin, Error bars indicate mean ± SD of 3 independent experiments. ***, p< 0.001. c Knockdown of SPI1 inhibited cell proliferation on the basis of CCK8 assays. Error bars represent the mean ± SD of 5 independent experiments. **, p< 0.01;***, p< 0.001. d Inhibition of SPI1 decreased cell migration as determined by transwell assays. The bar chart represents the migration cell numbers. Error bars represent the mean ± SD of 5 different field. ***, p< 0.001. e Inhibition of SPI1 decreased cell migration as determined by wound healing assay. The bar chart represents the percentage of distance at 24h or 48h divided by the distance at 0h. Date are presented as mean ± SD of 3 independent experiments. ***, p< 0.001

Fig. 3.

Knockdown of SPI1 in glioma cells inhibit cell cycle and promote apoptosis in vitro. a Cell cycle was analyzed by flow cytometry after knocking down PAICS. The results showed that PAICS depletion led G1 arrest. b Cell apoptosis was analyzed by flow cytometry after knocking down PAICS. The results showed that PAICS depletion leading to an increased rate of apoptosis

Fig. 4.

SPI1 binds to the promoter regions of PAICS and regulates PAICS expression. a Schematic of the PAICS promoter luciferase construct is depicted with the locations of the GGGAAG element and the sequences of mutation. Relative luciferase activities of the Wt or Mut GGGAAG regions in U87 and U251 cells were examined after transfecting with SPI1 48 hour later. The values of PGL3-WT and PGL3-Mut were relative to PGL3-basic. Data are mean ± SD for triplicate samples. ***, p < 0.001. b SPI1 binding at the promoter region of PAICS containing the GGGAAG element was assessed by CHIP assay. c PAICS RNA levels after transfecting the SPI1 interference fragment into the U87 and U251 cells. PAICS expression was normalized to GAPDH. Error bars indicate mean ±SD of 3 independent experiments. *, p < 0.05; ** p < 0.01; ***, p < 0.001

Fig. 5.

SPI1 promotes glioma cell proliferation and migration through PAICS. a PAICS can reverse the reduction of cell proliferation induced by SPI1 interference on the basis of CCK8 assays. Error bars represent the mean ± SD of 3 independent experiments. **, p< 0.01;***, p< 0.001. b PAICS can reverse the reduction of cell migration induced by SPI1 interference on the basis of transwell assays. The bar chart represents the migration cell numbers. Error bars represent the mean ± SD of 3 different field. ***, p< 0.001