PDGF-D Promotes Epithelial-Mesenchymal Transition of Glioma Cells Through the NF-κB/NOTCH1 Pathway

Abstract

Background: Platelet-derived growth factor-D (PDGF-D) is expressed at high levels in various tumors and is involved in epithelial-mesenchymal transition (EMT) and the malignant behavior of cancer cells. However, its role in glioma progression and the underlying molecular mechanisms remain unclear.

Methods: We used data from the Chinese Glioma Genome Atlas to evaluate the correlation among PDGF-D expression, tumor grade, and phenotype of glioma. The in situ expression of PDGF-D in clinical glioma specimens was analyzed through immunohistochemistry. Colony formation assays and transwell assays were performed for functional evaluation of glioma cell lines with PDGF-D knockdown or overexpression. Western blotting and RT-qPCR were conducted to explore molecular mechanisms.

Results: PDGF-D was significantly upregulated in high-grade glioma and was associated with the malignant phenotype and poor prognosis. Knocking down PDGF-D in the LN18 glioma cell line reduced the expression of phosphorylated p65 and NOTCH1 and inhibited clonal proliferation, migration, invasion, and the EMT program. In contrast, inhibiting p65 phosphorylation in glioma cells overexpressing PDGF-D led to the downregulation of NOTCH1 and reversed EMT.

Conclusion: PDGF-D promotes the invasion and migration of glioma cells by activating the NF-κB/NOTCH1 pathway.

Keywords: EMT; NF‐κB; NOTCH1; PDGF‐D; glioma.

FIGURE 1

PDGF‐D was highly expressed in glioma and promoted proliferation. (A) CGGA data showing the correlation between PDGF‐D expression in glioma and the degree of malignancy and prognosis. (B, C) Representative immunostaining images showing in situ expression of PDGF‐D and PDGFR‐β in glioma tissues and normal brain tissues. The brown‐stained regions correspond to positive staining. Images are 400× magnified. (D) PDGF‐D and PDGFR‐β protein expression in the indicated glioma cell lines. (E, G) Changes in PDGF‐D mRNA and protein expression after stable transfection of PDGF‐D sh1 and PDGF‐D sh2 in LN18 cells. (F, H) Changes in PDGF‐D mRNA and protein expression levels in U87 cells overexpressing PDGF‐D. (I) Colonies formed by the control and PDGF‐D‐knockdown LN18 cells. (J) Colonies formed by the control and PDGF‐D‐overexpressing U87 cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2

PDGF‐D promoted the EMT of glioma cells. (A) The effect of PDGF‐D knockdown on the migration and invasion of LN18 cells. (B) The effect of PDGF‐D overexpression on the migration and invasion of U87 cells. (C, E) The effect of PDGF‐D knockdown on the expression of MMP‐2 and EMT‐related proteins in LN18 cells. (D, F) The effect of PDGF‐D upregulation on the expression of MMP‐2 and EMT‐related proteins in U87 cells. *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 3

PDGF‐D promoted EMT in glioma cells via NOTCH1. (A, C) Effect of PDGF‐D knockdown on NOTCH1 mRNA and NICD protein expression in LN18 cells. (B, D) Effect of PDGF‐D overexpression on NOTCH1 mRNA and NICD protein expression in U87 cells. (E) Effect of Dll4 on NICD protein expression in LN18 cells with PDGF‐D knockdown. (F) Effect of DAPT on NICD protein expression in U87 cells overexpressing PDGF‐D. (G) Effect of Dll4 on the migration and invasion of LN18 cells with PDGF‐D knockdown. (H) Effect of DAPT on migration and invasion of U87 cells overexpressing PDGF‐D. (I, K) Effect of Dll4 on the expression levels of MMP‐2 and EMT‐related proteins in LN18 cells with PDGF‐D knockdown. (J, L) Effect of DAPT on the expression levels of MMP‐2 and EMT‐related proteins in U87 cells overexpressing PDGF‐D. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. NC or OE‐Vec. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. PDGF‐D sh or OE‐PDGF‐D.

FIGURE 4

PDGF‐D upregulated NOTCH1 by upregulating the phosphorylation of p65. (A) Effect of PDGF‐D knockdown on the levels of p‐IκB and p‐p65 in LN18 cells. (B) Effect of PDGF‐D overexpression on the levels of p‐IκB and p‐p65 in U87 cells. (C) Effect of BAY 11–7082 on nuclear p‐p65 levels in U87 cells overexpressing PDGF‐D. (D, E) Effect of BAY 11–7082 on NOTCH1 mRNA and NICD protein expression in U87 cells overexpressing PDGF‐D. (F, G) Effect of BAY 11–7082 on the migration and invasion of U87 cells overexpressing PDGF‐D. (H, I) Effect of BAY 11–7082 on the expression levels of MMP‐2 and EMT‐related proteins in U87 cells overexpressing PDGF‐D. *p < 0.05, **p < 0.01, ***p < 0.001 vs. NC or OE‐Vec. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 vs. OE‐PDGF‐D.

FIGURE 5

Results of the in vivo experiment. (A) The tumor‐bearing nude mice were injected intraperitoneally with 2.5 mg/mL DAPT (100 mg/kg) or equal volume of physiological saline starting on the 8th day after subcutaneous inoculation of tumor cells, and on alternate days thereafter. The volume of subcutaneous tumors was measured every other day (B) Representative immunostaining images of tumor tissues showing expression of the indicated proteins. The brown‐colored regions are positively stained. Images are 200× magnified. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. OE‐Vec. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 vs. OE‐PDGF‐D.