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

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.

Figure 1. Anti-angiogenic property of miR-7 in vitro

(a) miR-7 inhibits HUVEC cell viability. Cell viability was measured at 72 hrs after transfection using MTS-read-out. Cells were transfected with increasing concentrations of miR-7, miR-scr or siPLK-1. siPLK-1 and miR-scr were used as positive and negative control. Data are normalized to cell viability of untreated cells and plotted as mean values ± s.d. (n=3). (b) Cellular miR-7 expression increases after transfection of HUVEC with increasing concentrations of miR-7. miR-7 expression was measured by stem-loop RT-PCR at 72 hrs after transfection. Data are normalized to untreated cells and plotted as mean values ± s.d. (n=3), * p<0.001. (c-d) miR-7 inhibits two-dimensional tube formation. HUVEC were transfected with 50 nM miR-7 or miR-scr and seeded on matrigel at 48hrs after transfection. Pictures were taken at 17 hrs after seeding (magnification in Supplementary Fig. S11). Two-dimensional tube-formation was quantified by counting number of branching points and calculating the cumulative length of the tube of each image. Data are plotted as mean values ± s.d. (n= 3), * p<0.0001. (e-f) miR-7 inhibits three-dimensional sprouting. HUVEC were transfected with 50 nM miR-7 or miR-scr. 24hrs after transfection cell-spheroids were embedded in a collagen matrix in the presence of basic Fibroblast Growth Factor (bFGF). Untreated HUVEC in the absence of bFGF were used as negative control and the sprouting of miR-7 and miR-scr treated cells were compared to the sprouting of bFGF activated HUVEC. Pictures were taken at 16 hrs after embedding. Three-dimensional sprouting was quantified by counting the sprouts and calculating the cumulative length of 10 individual spheroids for each treatment. Data are plotted as mean values ± s.d. (n= 10), * p<0.0001. (g-h) miR-7 inhibits migration. HUVEC were transfected with 50 nM miR-7 or miR-scr. Cells were harvested at 48hrs after transfection and equal amount of cells were seeded in 24-well plate and wounded by a scratch. Images were taken right after the wound scratch (T=0) and at 17hrs after scratching (T=17) Wound closure was quantified by calculating unclosed surface area relative to surface area right after the scratch wound. Data are plotted as mean values ± s.d. (n=3),* p<0.001.

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).

Figure 3. Effect of miR-7 on the CAM-assay

(a) Seed sequence of miR-7. Illustration of conserved seed sequence of miR-7 among different species. (b) miR-7 acts as vascular disrupting agent on CAM. Chick CAMs were treated locally within a nitrocellulose ring with 300 picomol miR-7 or miR-Scr using Lipofectamine 2000 or with 200 picomol sunitinib. Untreated and mock treated CAM were used as controls. Representative photographs were taken prior to transfection (T=0) and at 48 hrs after transfection (T=48).

Figure 4. Inhibitory effect of miR-7 on tumor growth by local delivery

(a) miR-7 inhibits tumor growth after local delivery. AJ mice bearing tumors with Neuro2A cells were treated locally with 10 μg miR-7 or 10 μg miR-Scr or PBS by intratumoral injection and electroporation. Arrows below the graph indicate treatment schedule (6 treatments, every other day). Data are plotted as mean values ± SEM (n=7), p<0.05. (b) Increased presence of miR-7 in miR-7 treated animals. The day after the last injection tumors were removed and total RNA was isolated. Delivery of miR-7 by electroporation into the tumor tissue was determined by stem loop RT-PCR. Values were normalized to U6 expression in the tumors. Data are plotted as mean values ± s.d. (n=3), *p<0.001. (c-d) miR-7 reduces angiogenesis in vivo after local delivery. Tumor sections were stained for CD31 (in brown) and microvessel density (MVD) was quantified by counting blood vessels in 6 random high magnification fields in each sample. Data are plotted as mean values ± s.d. (n=4), *p< 0.01. (e-f) miR-7 does not affect proliferation in Neuro2A tumors. The effect of miR-7 on tumor cell proliferation was determined by Ki-67 staining (in brown). Quantification of the images was performed in the same way as described in (c-d), and expressed as percentage of the PBS treated group. Data are plotted as mean values ± s.d. (n=4). Magnification of Fig 4c and 4e in Supplementary Fig. S12.

Figure 5. Inhibitory effect of miR-7 on tumor growth by systemic delivery

(a) miR-7 treated animals show pale and less vascularized tumors. Macroscopic images of the tumors after the 7^th dose administration. (b) miR-7 inhibits tumor growth after systemic delivery. Athymic Nude-Foxn1^nu mice bearing U-87 MG tumors were injected intravenously with αvβ3/αvβ5 targeted miR-7 nanoparticles (3 mg/kg miRNA). Arrows in the graph indicate the days of treatment (8 treatments, every other day). miR-7 treated mice showed significant tumor growth inhibition compared to vehicle treated mice. Data are plotted as mean values ± SEM (n=10), *p<0.05. (c-d) miR-7 reduces angiogenesis after systemic delivery. Tumor sections were stained for CD31 (in brown) and quantified by counting CD31 positive staining area (pixels) in 6 random fields in each tumor. Data are plotted as mean values ± s.d. (n=5), *p<0.001. Magnification of Fig 5c in Supplementary Fig. S13. (e-f) miR-7 reduces cell-proliferation in U-87 MG tumors after systemic delivery. Anti-proliferative effect of systemically delivered miR-7 was determined by Ki-67 staining, indicated as brown spots. Quantification of Ki-67 was performed by counting of the stains in 6 random fields in each tumor section and proliferation was expressed as percentage of PBS treated mice. Data are plotted as mean values ± s.d. (n=5), *p<0.001. (g-h) miR-7 reduces OGT in U-87 MG tumors after systemic delivery. Tumor sections stained for OGT (brown nuclei and brownish cytoplasm) and quantified by counting of the stains in 6 random fields in each tumor section. Data are plotted as mean values ±s.d. (n=5), * p=0.0004.