Silencing of Insulin-like Growth Factor-binding Protein-2 in Human Glioblastoma Cells Reduces Both Invasiveness and Expression of Progression-associated Gene CD24

Abstract

Glioblastoma multiforme (GBM) is a malignant brain tumor characterized by rapid growth and extensive invasiveness. Overexpression of insulin-like growth factor-binding protein-2 (IGFBP-2) has been reported in GBM. However, it remains to be determined how IGFBP-2 is involved in the progression of GBM. We utilized short hairpin-RNA (shRNA) expression retroviral vectors to inactivate the IGFBP-2 gene permanently in two human GBM cell lines, U251 and YKG-1. The stable knockdown of IGFBP-2 resulted in decreased invasiveness, decreased saturation density of the cells in vitro, and decreased tumorigenicity in nude mice. Transcriptional profiling of both lines revealed several genes that were significantly down-regulated by inactivation of IGFBP-2. One such gene was CD24, which has been implicated in progression of various cancers. Indeed, CD24 was expressed in most GBM cases and the inactivation of CD24 in GBM cells suppressed cellular invasiveness, as was the case for IGFBP-2. Forced overexpression of CD24 led to increased invasiveness of both IGFBP-2-inactivated GBM cell lines and also A172, a human GBM cell line with low endogenous CD24. Further supporting the inter-relationship between IGFBP-2 and CD24, knockdown of IGFBP-2 suppressed the CD24 promoter activity. Moreover, both CD24 promoter activity and in vitro invasiveness were restored in knockdown cells by transfection with an IGFBP-2 expression plasmid. These results indicate that CD24 is modulated by IGFBP-2 and contributes to IGFBP-2-enhanced invasiveness of GBM cells.

Hepatocyte Growth Factor Activator Inhibitor Type 1 Regulates Epithelial to Mesenchymal Transition through Membrane-Bound Serine Proteinases

“…Total RNAs were extracted with the Trizol reagent (Life Technologies), followed by DNase I (Roche Applied Science) treatment and phenolchloroform-isoamylalcohol extraction. For reverse transcription-PCR (RT-PCR), 3 Ag of total RNA were reverse transcribed with a mixture of oligo(dT) and random primer and processed for each PCR reaction as described previously (23). The PCR products were analyzed by 1.5% agarose gel electrophoresis.…”

“…Knockdown of HAI-1/SPINT1. The knockdown vector was constructed by using a short hairpin RNA (shRNA) expression retroviral vector pSINsihU6 (TAKARA Bio) as described previously (23). The target sequence of HAI-1/SPINT1 gene was 5 ¶-GGGCAGGCATAGACTTGAAGG and scramble is 5 ¶-GGGACGGAGATTTCACGAGGA.…”

Role of Insulin-Like Growth Factor Binding Protein 2 in Lung Adenocarcinoma

“…27 Cells were plated at 7 ϫ 10 5 per well in 60-mm dishes and transfected in triplicate by using the FuGENE 6 Transfection Reagent according to the manufacturer's protocol (Roche Diagnostics, Inc., Indianapolis, IN). We established stable cell lines COR-L105, NCI-H522, and HOP62 overexpressing IGFBP-2 after 4 weeks of selection in 400 g/ml of neomycin.…”

Plasma IGFBP-2 levels predict clinical outcomes of patients with high-grade gliomas

“…22 Previous studies have shown that increased IGFBP-2 expression in a tumor promotes glioma cell migration and invasion through activation of the matrix metalloproteinase 2 protein and integrin pathway [23][24][25][26][27] and that this increased expression is associated with glioma malignancy and poor survival of glioma patients. 12,13,16,18,[28][29][30][31][32][33] Attenuation of IGFBP-2 by the tumor suppressor protein IIp45 or using small interfering RNA resulted in decreased cell motility and invasion.…”

“…12,13,16,18,[28][29][30][31][32][33] Attenuation of IGFBP-2 by the tumor suppressor protein IIp45 or using small interfering RNA resulted in decreased cell motility and invasion. 23,26 Recently, IGFBP-2 has been found to be a "driver" oncogene for glioma development and progression. 28 IGFBP-2 overexpression has been linked to PTEN (phosphatase and tensin homolog) deletion and results in Akt activation.…”