MiR-15b and miR-152 reduce glioma cell invasion and angiogenesis via NRP-2 and MMP-3

Abstract

We tested invasion and angiogenesis related mRNA expression and miRNA profiles of glioma. Genes with mRNA expression that changed significantly were selected to predict possible miRNAs that regulate mRNA expression, and were then matched with miRNA results. NRP-2 with the matching miRNA miR-15b, and MMP-3 with the matching miRNA miR-152 were selected for further study. Luciferase activity assay confirmed that miR-15b and miR-152 attenuate expression of NRP-2 and MMP-3 protein by binding to NRP-2 and MMP-3 transcript, respectively. In vitro invasion assay data showed that miR-15b and miR-152 significantly decreased 9L cell invasiveness. In vitro tube formation assay data showed that miR-15b reduced tube formation. A preliminary pathway study indicated that miR-15b and miR-152 deactivated the MEK-ERK pathway via NRP-2 and MMP-3 in 9L cells, respectively.

Figure. 1

A. Laser Capture Microdissection. B. miRNA expression in 9L and primary rat astrocyte coculture. C. Protein expression in co-cultured 9L and primary rat astrocyte. D. protein expression in co-cultured 9L and rat astrocyte cell line. E. Luciferase activity assay. miR-152 significantly decreased luciferase activity in Luc-NRP-2-UTR (wild-type) which suggest that miR-15b directly targets the 3′-UTR of NRP-2 mRNA. miR-152b suppressed >50% activity in Luc-MMP-3-UTR (wild-type) which suggest that miR-152 directly targets the 3′-UTR of MMP-3 mRNA. 9L: 9L glioma cells. 9L(Astro): 9L glioma cells co-cultured with rat astrocyte. Astro: Rat astrocyte Astro (9L): Rat astrocyte co-cultured with 9L cells.

Figure. 2. miR-15b and miR152 decreased NRP-2 and MMP-3 expression and deactivated MEK-ERK pathway in 9L cells

NRP-2 expression was significantly decreased by miR-15b mimic transfection; MMP-3 expression was significantly decreased by miR-152 mimic transfection. Co-transfection of miR-15b and miR-152 mimic significantly decreased both MMP-3 and NRP-2 expression. P-ERK expression was significantly decreased by overexpression of miR-15b or miR-152.

Figure 3. miR-15b and miR 152 decreased 9L cell invasiveness

miR-15b and miR-152 significantly decreased the number of invaded cells. Anti-miR miRNA inhibitor co-transfected with miRNA mimic reversed the inhibition of invasion caused by miR-15b or miR152 mimic. Compared to Cel-miR-67 control, miR15b and miR-152 inhibited 9L cell invasion by 49.5±3.7% and 75.8±3.5%. Combination of miR-15b and miR-152 significantly decreased 9L cell invasion by 78.0±3.8%. Compared to miR-15b, co-transfection of anti-miR-15b miRNA inhibitor and miR-15b mimic increased invasion by 3.04±0.23 fold. Compared to miR-152, co-transfection of anti-miR-152 miRNA inhibitor and miR-152 mimic increased invasion by 2.85±0.12 fold.

Figure. 4. miR-15b reduced tube formation in cultured endothelial cells and anti-miR-15b inhibitor reversed the inhibition of tube formation caused by miR-15b

Culture medium from miR-15b mimic transfected cells decreased MBEC capillary tube formation by 85.7±7.2%. MiR-152 mimic did not change capillary tube formation in MBECs. Cotransfection of miR-15b and miR-152 mimic significantly decreased capillary tube formation by 83.4.9±3.8%. Anti-miR-15b miRNA inhibitor reversed the inhibition of tube formation in MBECs caused by miR-15b mimic. Compared to miR-15b mimic transfection, the tube formation of miR145 mimic and inhibitor cotransfection increased by 6.24±0.29 fold.