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Systemic miRNA-7 delivery inhibits tumor angiogenesis and growth in murine xenograft glioblastoma.

Babae N, Bourajjaj M, Liu Y, Van Beijnum JR, Cerisoli F, Scaria PV, Verheul M, Van Berkel MP, Pieters EH, Van Haastert RJ, Yousefi A, Mastrobattista E, Storm G, Berezikov E, Cuppen E, Woodle M, Schaapveld RQ, Prevost GP, Griffioen AW, Van Noort PI, Schiffelers RM - Oncotarget (2014)

Bottom Line: Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration.Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib.Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7.

View Article: PubMed Central - PubMed

Affiliation: Utrecht Institute for Pharmaceutical Sciences, University Utrecht, Utrecht, the Netherlands. These authors contributed equally to this work.

ABSTRACT
Tumor-angiogenesis is the multi-factorial process of sprouting of endothelial cells (EC) into micro-vessels to provide tumor cells with nutrients and oxygen. To explore miRNAs as therapeutic angiogenesis-inhibitors, we performed a functional screen to identify miRNAs that are able to decrease EC viability. We identified miRNA-7 (miR-7) as a potent negative regulator of angiogenesis. Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration. Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib. Local administration of miR-7 in an in vivo murine neuroblastoma tumor model significantly inhibited angiogenesis and tumor growth. Finally, systemic administration of miR-7 using a novel integrin-targeted biodegradable polymeric nanoparticles that targets both EC and tumor cells, strongly reduced angiogenesis and tumor proliferation in mice with human glioblastoma xenografts. Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7. Our study provides a comprehensive validation of miR-7 as novel anti-angiogenic therapeutic miRNA that can be systemically delivered to both EC and tumor cells and offers promise for miR-7 as novel anti-tumor therapeutic.

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miR-7 modifies endothelial gene expression(a) miR-7 changes the expression of 2500 HUVEC genes after transfection. HUVEC were transfected with either miR-7 or miR-Scr and the transcriptome was quantified by RNA-Seq. All differentially expressed genes, which are statistically significant down- or upregulated compared to miR-Scr (p-value< 0.05) are plotted. (b) Majority of miR-7 predicted target genes are downregulated. Differentially expressed genes that are regulated by miR-7 and are a predicted target genes of miR-7 are plotted. (c) mRNA expression of OGT is downregulated after miR-7 transfection. HUVEC were transfected with increasing concentrations of miR-7 or miR-Scr. OGT expression was measured by RT-PCR at 48 hrs after transfection. Data are normalized to untreated cells and plotted as mean values ± s.d. (n=3), *p<0.001. (d) OGT protein is downregulated after miR-7 transfection. HUVEC were transfected with increasing concentration of miR-7 or miR-Scr. OGT expression was determined by Western Blot analysis at 48 hrs after transfection. Beta-Actin was used as an internal control. (e) OGT is a target gene of miR-7. Luciferase activities in Hela cells co-transfected with a luciferase- OGT 3′UTR plasmid containing either wildtype (WT) 3′UTR or mutated sequence and either miR-Scr or miR-7 at 24 hrs post transfection. Data are normalized to miR-Scr and plotted as mean values ± s.d. (n=3), *p<0.0002. (f) Alignment of miR-7 and OGT sequence. Mutations were generated on the potential target sequence (horizontally underlined).
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Figure 2: miR-7 modifies endothelial gene expression(a) miR-7 changes the expression of 2500 HUVEC genes after transfection. HUVEC were transfected with either miR-7 or miR-Scr and the transcriptome was quantified by RNA-Seq. All differentially expressed genes, which are statistically significant down- or upregulated compared to miR-Scr (p-value< 0.05) are plotted. (b) Majority of miR-7 predicted target genes are downregulated. Differentially expressed genes that are regulated by miR-7 and are a predicted target genes of miR-7 are plotted. (c) mRNA expression of OGT is downregulated after miR-7 transfection. HUVEC were transfected with increasing concentrations of miR-7 or miR-Scr. OGT expression was measured by RT-PCR at 48 hrs after transfection. Data are normalized to untreated cells and plotted as mean values ± s.d. (n=3), *p<0.001. (d) OGT protein is downregulated after miR-7 transfection. HUVEC were transfected with increasing concentration of miR-7 or miR-Scr. OGT expression was determined by Western Blot analysis at 48 hrs after transfection. Beta-Actin was used as an internal control. (e) OGT is a target gene of miR-7. Luciferase activities in Hela cells co-transfected with a luciferase- OGT 3′UTR plasmid containing either wildtype (WT) 3′UTR or mutated sequence and either miR-Scr or miR-7 at 24 hrs post transfection. Data are normalized to miR-Scr and plotted as mean values ± s.d. (n=3), *p<0.0002. (f) Alignment of miR-7 and OGT sequence. Mutations were generated on the potential target sequence (horizontally underlined).

Mentions: One of the hallmarks of miRNAs is their ability to target and regulate multiple genes. To explain the anti-angiogenic properties of miR-7, gene expression analysis was performed using RNA-seq. Differential expression analysis showed that 2500 genes were significantly up- or downregulated after transfection of HUVEC with miR-7 mimic (p-value < 0.05, compared to miR-Scr, Fig. 2a). The majority, 1317, of the miR-7 modulated genes were down-regulated while 1183 were upregulated. Comparison of the list of down-regulated genes with a list of 11 clinically explored molecular targets in angiogenesis (Table 2) showed that eight genes matched the clinical target list. For each of these “therapeutic targets”, compounds are presently in clinical development (Table 2). The complete gene expression profile was analyzed using Ingenuity Pathway Analysis software (IPA). First, the set of regulated genes was overlaid with the 1051 predicted miR-7 targets, based on the TargetScan prediction tool in IPA. In total 282 predicted miR-7 targets were up- or downregulated within the set of 2500 genes (Fig. 2b). Consistent with the mechanism that miRNA down-regulate mRNA expression, 264 predicted targets (94%) were downregulated while only 18 (6%) were upregulated.


Systemic miRNA-7 delivery inhibits tumor angiogenesis and growth in murine xenograft glioblastoma.

Babae N, Bourajjaj M, Liu Y, Van Beijnum JR, Cerisoli F, Scaria PV, Verheul M, Van Berkel MP, Pieters EH, Van Haastert RJ, Yousefi A, Mastrobattista E, Storm G, Berezikov E, Cuppen E, Woodle M, Schaapveld RQ, Prevost GP, Griffioen AW, Van Noort PI, Schiffelers RM - Oncotarget (2014)

miR-7 modifies endothelial gene expression(a) miR-7 changes the expression of 2500 HUVEC genes after transfection. HUVEC were transfected with either miR-7 or miR-Scr and the transcriptome was quantified by RNA-Seq. All differentially expressed genes, which are statistically significant down- or upregulated compared to miR-Scr (p-value< 0.05) are plotted. (b) Majority of miR-7 predicted target genes are downregulated. Differentially expressed genes that are regulated by miR-7 and are a predicted target genes of miR-7 are plotted. (c) mRNA expression of OGT is downregulated after miR-7 transfection. HUVEC were transfected with increasing concentrations of miR-7 or miR-Scr. OGT expression was measured by RT-PCR at 48 hrs after transfection. Data are normalized to untreated cells and plotted as mean values ± s.d. (n=3), *p<0.001. (d) OGT protein is downregulated after miR-7 transfection. HUVEC were transfected with increasing concentration of miR-7 or miR-Scr. OGT expression was determined by Western Blot analysis at 48 hrs after transfection. Beta-Actin was used as an internal control. (e) OGT is a target gene of miR-7. Luciferase activities in Hela cells co-transfected with a luciferase- OGT 3′UTR plasmid containing either wildtype (WT) 3′UTR or mutated sequence and either miR-Scr or miR-7 at 24 hrs post transfection. Data are normalized to miR-Scr and plotted as mean values ± s.d. (n=3), *p<0.0002. (f) Alignment of miR-7 and OGT sequence. Mutations were generated on the potential target sequence (horizontally underlined).
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Related In: Results  -  Collection

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Figure 2: miR-7 modifies endothelial gene expression(a) miR-7 changes the expression of 2500 HUVEC genes after transfection. HUVEC were transfected with either miR-7 or miR-Scr and the transcriptome was quantified by RNA-Seq. All differentially expressed genes, which are statistically significant down- or upregulated compared to miR-Scr (p-value< 0.05) are plotted. (b) Majority of miR-7 predicted target genes are downregulated. Differentially expressed genes that are regulated by miR-7 and are a predicted target genes of miR-7 are plotted. (c) mRNA expression of OGT is downregulated after miR-7 transfection. HUVEC were transfected with increasing concentrations of miR-7 or miR-Scr. OGT expression was measured by RT-PCR at 48 hrs after transfection. Data are normalized to untreated cells and plotted as mean values ± s.d. (n=3), *p<0.001. (d) OGT protein is downregulated after miR-7 transfection. HUVEC were transfected with increasing concentration of miR-7 or miR-Scr. OGT expression was determined by Western Blot analysis at 48 hrs after transfection. Beta-Actin was used as an internal control. (e) OGT is a target gene of miR-7. Luciferase activities in Hela cells co-transfected with a luciferase- OGT 3′UTR plasmid containing either wildtype (WT) 3′UTR or mutated sequence and either miR-Scr or miR-7 at 24 hrs post transfection. Data are normalized to miR-Scr and plotted as mean values ± s.d. (n=3), *p<0.0002. (f) Alignment of miR-7 and OGT sequence. Mutations were generated on the potential target sequence (horizontally underlined).
Mentions: One of the hallmarks of miRNAs is their ability to target and regulate multiple genes. To explain the anti-angiogenic properties of miR-7, gene expression analysis was performed using RNA-seq. Differential expression analysis showed that 2500 genes were significantly up- or downregulated after transfection of HUVEC with miR-7 mimic (p-value < 0.05, compared to miR-Scr, Fig. 2a). The majority, 1317, of the miR-7 modulated genes were down-regulated while 1183 were upregulated. Comparison of the list of down-regulated genes with a list of 11 clinically explored molecular targets in angiogenesis (Table 2) showed that eight genes matched the clinical target list. For each of these “therapeutic targets”, compounds are presently in clinical development (Table 2). The complete gene expression profile was analyzed using Ingenuity Pathway Analysis software (IPA). First, the set of regulated genes was overlaid with the 1051 predicted miR-7 targets, based on the TargetScan prediction tool in IPA. In total 282 predicted miR-7 targets were up- or downregulated within the set of 2500 genes (Fig. 2b). Consistent with the mechanism that miRNA down-regulate mRNA expression, 264 predicted targets (94%) were downregulated while only 18 (6%) were upregulated.

Bottom Line: Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration.Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib.Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7.

View Article: PubMed Central - PubMed

Affiliation: Utrecht Institute for Pharmaceutical Sciences, University Utrecht, Utrecht, the Netherlands. These authors contributed equally to this work.

ABSTRACT
Tumor-angiogenesis is the multi-factorial process of sprouting of endothelial cells (EC) into micro-vessels to provide tumor cells with nutrients and oxygen. To explore miRNAs as therapeutic angiogenesis-inhibitors, we performed a functional screen to identify miRNAs that are able to decrease EC viability. We identified miRNA-7 (miR-7) as a potent negative regulator of angiogenesis. Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration. Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib. Local administration of miR-7 in an in vivo murine neuroblastoma tumor model significantly inhibited angiogenesis and tumor growth. Finally, systemic administration of miR-7 using a novel integrin-targeted biodegradable polymeric nanoparticles that targets both EC and tumor cells, strongly reduced angiogenesis and tumor proliferation in mice with human glioblastoma xenografts. Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7. Our study provides a comprehensive validation of miR-7 as novel anti-angiogenic therapeutic miRNA that can be systemically delivered to both EC and tumor cells and offers promise for miR-7 as novel anti-tumor therapeutic.

Show MeSH
Related in: MedlinePlus