To investigate the regulating mechanism of Notch1 signaling pathway on the invasion and migration of glioma initiating cells (GICs).

(1) Box-plotting was conducted to analyze the mRNA expression of Notch1 in normal brain tissue and glioblastoma tissue using Bredel Brain, Sun Brain and TCGA databases; Kaplan-Meier survival analysis was conducted to analyze the association between the prognosis of glioma patients with the expression of Hes1 in TCGA database; Heatmap was conducted to analyze the expression of Notch1 and CXCR4 in GICs and common cell line in GEO database. (2) Magnetic activated cell sorting was adopted to establish cell lines of U87 GICs and U251 GICs; immunofluorescence staining was used to detect expression of CXCR4 and Notch1. After the cell lines of U87 GICs and U251 GICs were divided into DMSO, shNC, MK0752 and shNotch1 groups, the shNotch1 and shNC groups were transfected respectively with recombinant lentivirus of Notch1-shRNA and its control sequence while the MK0752 and DMSO groups were added respectively with MK-0752 of 80 nmol/mL and the same amount of DMSO. The protein expression of Notch1, CXCR4 and p-mTOR was detected by Western blotting in the 4 groups. The capabilities of invasion and migration of the GICs were detected by Transwell assay in the shNotch1 and shNC groups.

(1) The box-plotting showed the mRNA expression of Notch1 in the glioblastoma tissue was significantly higher than in the normal brain tissue (P<0.05). The Kaplan-Meier survival analysis showed that the life span of glioma patients with high expression of Hes1 was significantly shorter than that of those with low expression of Hes1 (P<0.05). Heatmaps showed that the expression levels of Notch1 and CXCR4 in GICs were higher than in the common cell line. (2) The immunofluorescence staining showed that Notch1 and CXCR4 were highly expressed and colocalized in cell lines of U87 GICs and U251 GICs. The Western blotting showed that the protein expression of Notch1, CXCR4 and p-mTOR in the cell lines of U87 GICs and U251 GICs in the MK0752 and shNotch1 groups was lower than that in the DMSO and shNC groups. Transwell assay showed that the penetrating-membrane cells per visual field in the shNotch1 group were significantly fewer than those in the shNC group (P<0.05).

Notch1 signaling pathway can promote invasion and migration of GICs through regulating CXCR4 expression.

Notch1 pathway; CXCR4; Glioma initiating cells; Invasion; Migration