Presenilin1 exerts antiproliferative effects by repressing the Wnt/β-catenin pathway in glioblastoma

Abstract

Background: Glioblastoma and Alzheimer's disease (AD) are the most common and devastating diseases in the central nervous system. The dysfunction of Presenilin1 is the main reason for AD pathogenesis. However, the molecular function of Presenilin1 and its relative mechanism in glioblastoma remain unclear.

Methods: Expression of presenilin1 in glioma was determined by IHC. CCK-8, colony formation, Flow cytometry, Edu staining were utilized to evaluate functions of presenilin1 on glioblastoma proliferation. The mechanism of above process was assessed by Western blotting and cell immunofluorescence. Mouse transplanting glioblastoma model and micro-MRI detection were used to verified presenilin1 function in vivo.

Results: In this study, we found that all grades of glioma maintained relatively low Presenilin1 expression and that the expression of Presenilin1 in high-grade glioma was significantly lower than that in low-grade glioma. Moreover, the Presenilin1 level had a positive correlation with glioma and glioblastoma patient prognosis. Next, we determined that Presenilin1 inhibited the growth and proliferation of glioblastoma cells by downregulating CDK6, C-myc and Cyclin D1 to arrest the cell cycle at the G1/S phase. Mechanistically, Presenilin1 promoted the direct phosphorylation of β-catenin at the 45 site and indirect phosphorylation at the 33/37/41 site, then decreased the stabilized part of β-catenin and hindered its translocation from the cytoplasm to the nucleus. Furthermore, we found that Presenilin1 downregulation clearly accelerated the growth of subcutaneous glioblastoma, and Presenilin1 overexpression significantly repressed the subcutaneous and intracranial transplantation of glioblastoma by hindering β-catenin-dependent cell proliferation.

Conclusion: Our data implicate the antiproliferative effect of Presenilin1 in glioblastoma by suppressing Wnt/β-catenin signaling, which may provide a novel therapeutic agent for glioblastoma. Video Abstract.

Keywords: Cell cycle; Glioblastoma; Presenilin1; Proliferation.

Fig. 1

a Representative IHC staining of Presenilin1 protein in low grade glioma tissues (LGG, left panels) and high grade glioma tissues (HGG, right panels). Scale bars = 50 um and 20 um respectively. b Statistical analysis of the relative expression levels of Presenilin1 in LGG and HGG, * p < 0.05. c Kaplan-Meier analysis for all grade glioma patients, patients in the high Presenilin1 group (n = 122) and in the low Presenilin1 group (n = 89) (P = 0.0000, log-rank test). d Kaplan-Meier analysis for glioma patients. Patients in the high Presenilin1 group (n = 28) and in the low Presenilin1 group (n = 91) (P = 0.0257, log-rank test). e Kaplan-Meier analysis for all grade glioma patients. Patients in the high Presenilin1 group (n = 180) and in the low Presenilin1 group (n = 92) (P = 0.0019, log-rank test). f U87 cells were transfected with lentivirus contain sh-RNA target Presenilin1(sh-PS1) or full-length gene of Presenilin1(Lv-PS1).Sh-NC and Lv-NC were used as control, then Western-blot to detect the down- and over-expression effect of lentivirus in U87 cells, *p < 0.05,**p < 0.01

Fig. 2

a CCK-8 assays were performed in U87 cell when loss- or gain- function of Presenilin1, OD value were detected at day 1,2,3,5 and 7 and were showed as mean ± SD. b U251 cells down- or up-expressed of Presenilin1, then were used for CCK-8 assays to reveal the function of Presenilin1 in U251 cells. c Clone formation of the U87 cells after knockdown or over-expression of Presenilin1, histograms showing the clone formation in each group, Scale bars = 1 cm. d Representative image of clone formation in U251 cells after Sh-Presenilin1 or Lv-Presenilin1 treatment, Scale bars = 1 cm.* p < 0.05,**p < 0.01

Fig. 3

a U87 and U251 cells were over-expressed of Presenilin1, then were used to flow cytometry assays to analyse the cell cycle. Histograms showing the percent of cell at G1, S and G2 phase. b Flow cytometry assays were applied to investigate the influence of down expression of Presenilin1 on cell cycle in U87 and U251 cells. c Edu staining to show the proliferation states of U87 and U251 cells after Presenilin1 were up-regulated, histograms showing the Edu positive cell ratio in each groups, Scale bars = 50um. d Representative Edu images and histograms to show the influence of Presenilin1 repression on glioma proliferation, Scale bars = 50um. e Western-blot assays to show the expression of C-myc, CDK6 and CyclinD1 when Presenilin1 down- or up-regulation in U87and U251 cell. * p < 0.05,** p < 0.01

Fig. 4

a-c the correlation of Presenilin1 with β-catenin, CyclinD1 and C-myc from French-284-glioma dataset. d Western-blot assays to investigate the expression levels of p45-β-catenin, p33/37/41-β-catenin, total β-catenin, p9-GSK-3β and GSK-3β in U87 cells after down- or up-expression of Presenilin1. e-f The expression of β-catenin was detected by IF assays in U87 and U251 cells after down- or up-expression of Presenilin1. the co-localization mask and Pearson’s index were analyzed by Image J. scale bar = 20um, * p < 0.05,** p < 0.01

Fig. 5

a-b Subcutaneous transplanting gliomas were utilized to analyse the function of Presenilin1 on glioma in vivo, tumor values were measured every-3 days and calculated to histogram. c Representative IHC images of Ki67 expression in 4 groups of subcutaneous glioma tissues, scale bar = 50um. d The expression of p45-β-catenin, p33/37/41-β-catenin and total β-catenin in 4 groups of subcutaneous glioma tissues were detected by IHC, scale bar = 50um. * p < 0.05,** p < 0.01. e Western-blot assays to investigate the expression levels of p45-β-catenin, p33/37/41-β-catenin, total β-catenin, p9-GSK-3β and GSK-3β in U87 cells after down- or up-expression of PSEN1

Fig. 6

a-d Intracranial xenografts of gliomas were established with GL2161 cells after up-regulation of Presenilin1, representative MRI image showing the the tumor size at 7 days and 19 days after transplating in each groups, scale bar = 5 mm. Histograms showing the tumor values at 7 days and 19 days after transplating in each groups. e The representative gross image of glioma after dissected from mice in each group. f IHC staining of Ki-67 to show the proliferation state of transplating glioma, Scale bar = 50um. g The survival curve of glioma-bearing mice was analyzed by Kaplan-Meier. h The schematic model: Presenilin1 exert anti-glioma function by increasing the phosphorylation of β-catenin and degradation, then arrest cell cycle at G1/S phase.* p < 0.05,** p < 0.01