RBM8A Promotes Glioblastoma Growth and Invasion Through the Notch/STAT3 Pathway

Abstract

Background: Glioblastoma (GBM) is a prevalent brain malignancy with an extremely poor prognosis, which is attributable to its invasive biological behavior. The RNA-binding motif protein 8A (RBM8A) has different effects on various human cancers. However, the role of RBM8A in GBM progression remains unclear.

Methods: We investigated the expression levels of RBM8A in 94 GBM patients and explored the correlation between RBM8A expression and patient prognosis. Using in vitro and in vivo assays, combined with GBM sequencing data from the Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA), we examined whether and how RBM8A contributes to GBM progression.

Results: RBM8A was up-regulated in GBM tissues, and its higher expression correlated with worse prognosis. Knockdown of RBM8A inhibited GBM progression and invasion ability both in vitro and in vivo. On the contrary, overexpression of RBM8A promoted GBM progression and invasion ability. Enrichment analysis of differentially expressed genes in GBM data identified the Notch1/STAT3 network as a potential downstream target of RBM8A, and this was supported by molecular docking studies. Furthermore, we demonstrated that RBM8A regulates the transcriptional activity of CBF1. The γ-secretase inhibitor DAPT significantly reversed RBM8A-enhanced GBM cell proliferation and invasion, and was associated with down-regulation of p-STAT3 and Notch1 protein. Finally, the gene set variance analysis score of genes involved in regulation of the Notch1/STAT3 network by RBM8A showed good diagnostic and prognostic value for GBM.

Conclusions: RBM8A may promote GBM cell proliferation and migration by activating the Notch/STAT3 pathway in GBM cells, suggesting that RBM8A may serve as a potential therapeutic target for the treatment of GBM.

Keywords: Notch; RBM8A; glioblastoma; prognosis; stat3.

Figure 1

Up-regulated RBM8A expression levels suggest an unfavorable prognosis in GBM. (A) RBM8A was up-regulated in GBM based on The Cancer Genome Atlas. (B) Kaplan-Meier curves for the GBM samples in TCGA based on optimal gene expression grouping. GBM patients with high expression of RBM8A showed poor prognosis. (C) Representative images from immunohistochemical staining of tumor sections from GBM patients. Immunoglobulin (IgG) was used as a negative control. Magnification: 40× or 400×. Representative photographs show low or high RBM8A expression in GBM tissues. (D) Kaplan-Meier curves of overall survival for patients whose tumors showed low or high RBM8A expression.

Figure 2

Effects of RBM8A knockdown on proliferation of U87-MG and U251-MG cells and their invasive activity. (A) Expression of RBM8A in U87 or U251-MG cells transformed with control lentivirus (NC) or lentivirus expressing the knockdown-1 (KD1) or knockdown-2 (KD2) small hairpin RNA against the RBM8A gene were determined by western-blotting. (B) Cell proliferation was analyzed using the CCK8 assay as indicated. (C) Cell proliferation was also measured using the EdU assay. Representative fluorescence micrographs of the different cell cultures are shown. Immunostaining levels are quantified in the plots below. (D) Transwell analysis of migration, using two-chamber wells without a Matrigel-coated insert. Magnification, 200×. (E) Transwell analysis of invasion, using two-chamber wells with a Matrigel-coated insert. All micrographs are shown at 200× magnification.

Figure 3

Effects of RBM8A overexpression on proliferation of A172 and T98G GBM cells and their invasive activity. (A) Expression of RBM8A in T98G cells transformed with control lentivirus (NC) or lentivirus expressing RBM8A (RBM8A-OE) was assessed by western blotting. (B) Cell proliferation was analyzed using the CCK8 assay as indicated. (C) Cell proliferation was also measured using the EdU assay. Representative fluorescence micrographs of the different cell cultures are shown. (D) Transwell analysis with or without RBM8A overexpression. (E) Matrigel-Transwell analysis with or without RBM8A overexpression. All micrographs are shown at 200× magnification.

Figure 4

Effects of RBM8A expression on the Notch/STAT3 pathway in GBM cells. (A) Quadrant plot. The genes in quadrant 2 were upregulated genes consistent with RBM8A expression. (B) Analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed on the consistently up-regulated genes. (C) RBM8A affects biological pathways by regulating Notch1 and STAT3. (D) Molecular docking of RBM8A and CBF1. (E) Western blot analysis of Notch1, phosphorylated STAT3 (p-STAT3), total STAT3 (STAT3), phosphorylated H3 (p-H3), total H3 (H3), and actin in U87-MG and U251-MG cells transformed with negative control (NC) lentivirus or with lentivirus encoding RBM8A-KD1 or -KD2. (F) Western blot analysis of the same proteins in A172 and T98G cells transformed with NC lentivirus or lentivirus encoding RBM8A (RBM8A-OE). (G) Luciferase reporter assay of RBM8A-induced activation of CBF1 in GBM cells. (H) RBM8A regulates the Notch/STAT3 signaling pathway by targeting CBF1, which may be a mechanism by which RBM8A contributes to GBM development.

Figure 5

Involvement of the Notch/STAT3 pathway in RBM8A-mediated GBM cell proliferation and invasion. (A) Cell proliferation were analyzed in A172 or T98G cells overexpressing RBM8A (OE) in the presence or absence of the γ-secretase inhibitor DAPT using the CCK8 assay. (B) Transwell analysis with or without DAPT. (C) Matrigel-Transwell analysis with or without DAPT. (D) Western blot analysis of Notch1, phospho-STAT3, total STAT3, and actin after incubation with or without DAPT. All micrographs are shown at a magnification of 200×.

Figure 6

Effects of RBM8A reduction on GBM progression in vivo. (A) Nude mice were intracranially injected with U87-MG cells transformed with empty lentivirus (NC) or lentivirus encoding short hairpin RBM8A-KD1. At 6 weeks after injection, animals were examined by MRI. (B) Representative photomicrographs of tumor sections stained with hematoxylin-eosin (H&E). (C) Immunohistochemistry of tumor tissues stained with antibody against RBM8A. (D) Immunohistochemistry of tumor tissues stained with antibody against Notch1.