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MicroRNA-21 exhibits antiangiogenic function by targeting RhoB expression in endothelial cells.

Sabatel C, Malvaux L, Bovy N, Deroanne C, Lambert V, Gonzalez ML, Colige A, Rakic JM, Noël A, Martial JA, Struman I - PLoS ONE (2011)

Bottom Line: Expression of miR-21 in endothelial cells also led to a reduction in the organization of actin into stress fibers, which may explain the decrease in cell migration.Further mechanistic studies showed that miR-21 targets RhoB, as revealed by a decrease in RhoB expression and activity in miR-21 overexpressing cells.Our results identify miR-21 as a new angiogenesis inhibitor and suggest that inhibition of cell migration and tubulogenesis is mediated through repression of RhoB.

View Article: PubMed Central - PubMed

Affiliation: Unit of Molecular Biology and Genetic Engineering, GIGA-Research, University of Liège, Sart Tilman, Liège, Belgium.

ABSTRACT

Background: MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that regulate gene expression at post-transcriptional level. The recent discovery of the involvement of these RNAs in the control of angiogenesis renders them very attractive in the development of new approaches for restoring the angiogenic balance. Whereas miRNA-21 has been demonstrated to be highly expressed in endothelial cells, the potential function of this miRNA in angiogenesis has never been investigated.

Methodology/principal findings: We first observed in endothelial cells a negative regulation of miR-21 expression by serum and bFGF, two pro-angiogenic factors. Then using in vitro angiogenic assays, we observed that miR-21 acts as a negative modulator of angiogenesis. miR-21 overexpression reduced endothelial cell proliferation, migration and the ability of these cells to form tubes whereas miR-21 inhibition using a LNA-anti-miR led to opposite effects. Expression of miR-21 in endothelial cells also led to a reduction in the organization of actin into stress fibers, which may explain the decrease in cell migration. Further mechanistic studies showed that miR-21 targets RhoB, as revealed by a decrease in RhoB expression and activity in miR-21 overexpressing cells. RhoB silencing impairs endothelial cell migration and tubulogenesis, thus providing a possible mechanism for miR-21 to inhibit angiogenesis. Finally, the therapeutic potential of miR-21 as an angiogenesis inhibitor was demonstrated in vivo in a mouse model of choroidal neovascularization.

Conclusions/significance: Our results identify miR-21 as a new angiogenesis inhibitor and suggest that inhibition of cell migration and tubulogenesis is mediated through repression of RhoB.

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RhoB-silencing alters endothelial cell migration, proliferation and tubulogenesis.A. Predicted miR-21 binding site within the RhoB 3′UTR. B. The wild-type (WT RhoB 3′UTR) or mutated (Mut RhoB 3′UTR) reporter plasmid was cotransfected into HEK293T cells with a precursor of miR-21 (Pre-21) or with a precursor control (Pre-Ctrl). Luciferase activities were quantified 48 h after transfection as described in the materials and methods section (Luciferase reporter assay and cloning). Renilla luciferase activity was normalized by Firefly luciferase activity. C. HUVECs were transfected with non-silencing siRNA (siRNA-Ctrl) or with RhoB siRNA and the RhoB protein level was measured after 48 h by Western blotting. The ERK1/2 level was also measured as an internal control. D–H. HUVECs were transfected as described above and were assessed for migration, tubulogenesis and proliferation after 48 h. D–E. Migration of transfected HUVECs in a scratch-wound assay 16 h after treatment with bFGF (10 ng/ml) and VEGFa (50 ng/ml) (n = 6–12 measurements/condition; n = 3 experiments). F–G. Transfected HUVECs were seeded onto Matrigel in EGM-2 and then allowed to form capillary-like structures for 16 h. Living cells were labeled with calcein-AM. Representative figures are shown in (F). Branching numbers (G) were quantified with Image J software (n = 5–10 pictures/condition; n = 3 experiments). H. Proliferation was assayed in transfected HUVEC treated with bFGF (10 ng/ml) and VEGFa (50 ng/ml) by measuring BrdU incorporation (n = 3). Data are means with the SD. *p<0.05 versus corresponding control.
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pone-0016979-g005: RhoB-silencing alters endothelial cell migration, proliferation and tubulogenesis.A. Predicted miR-21 binding site within the RhoB 3′UTR. B. The wild-type (WT RhoB 3′UTR) or mutated (Mut RhoB 3′UTR) reporter plasmid was cotransfected into HEK293T cells with a precursor of miR-21 (Pre-21) or with a precursor control (Pre-Ctrl). Luciferase activities were quantified 48 h after transfection as described in the materials and methods section (Luciferase reporter assay and cloning). Renilla luciferase activity was normalized by Firefly luciferase activity. C. HUVECs were transfected with non-silencing siRNA (siRNA-Ctrl) or with RhoB siRNA and the RhoB protein level was measured after 48 h by Western blotting. The ERK1/2 level was also measured as an internal control. D–H. HUVECs were transfected as described above and were assessed for migration, tubulogenesis and proliferation after 48 h. D–E. Migration of transfected HUVECs in a scratch-wound assay 16 h after treatment with bFGF (10 ng/ml) and VEGFa (50 ng/ml) (n = 6–12 measurements/condition; n = 3 experiments). F–G. Transfected HUVECs were seeded onto Matrigel in EGM-2 and then allowed to form capillary-like structures for 16 h. Living cells were labeled with calcein-AM. Representative figures are shown in (F). Branching numbers (G) were quantified with Image J software (n = 5–10 pictures/condition; n = 3 experiments). H. Proliferation was assayed in transfected HUVEC treated with bFGF (10 ng/ml) and VEGFa (50 ng/ml) by measuring BrdU incorporation (n = 3). Data are means with the SD. *p<0.05 versus corresponding control.

Mentions: To confirm that RhoB is a direct target of miR-21, we constructed a luciferase reporter vector encoding the complete 3′UTR of RhoB (WT RhoB 3′UTR) as well as a control vector containing mismatches in predicted miR-21 binding site (Mut RhoB 3′UTR) (Figure 5A). Cotransfection of the WT RhoB 3′UTR plasmid and pre-miR-21 in HEK-293T cells resulted in a strong decrease in luciferase activity suggesting that RhoB mRNA is a direct target of miR-21. Importantly, mutations in the sequence targeted by miR-21 in RhoB 3′UTR reduced the observed down-regulation of luciferase activity by pre-miR-21 validating that this predicted binding site is necessary for miR-21 dependent RhoB expression (Figure 5B).


MicroRNA-21 exhibits antiangiogenic function by targeting RhoB expression in endothelial cells.

Sabatel C, Malvaux L, Bovy N, Deroanne C, Lambert V, Gonzalez ML, Colige A, Rakic JM, Noël A, Martial JA, Struman I - PLoS ONE (2011)

RhoB-silencing alters endothelial cell migration, proliferation and tubulogenesis.A. Predicted miR-21 binding site within the RhoB 3′UTR. B. The wild-type (WT RhoB 3′UTR) or mutated (Mut RhoB 3′UTR) reporter plasmid was cotransfected into HEK293T cells with a precursor of miR-21 (Pre-21) or with a precursor control (Pre-Ctrl). Luciferase activities were quantified 48 h after transfection as described in the materials and methods section (Luciferase reporter assay and cloning). Renilla luciferase activity was normalized by Firefly luciferase activity. C. HUVECs were transfected with non-silencing siRNA (siRNA-Ctrl) or with RhoB siRNA and the RhoB protein level was measured after 48 h by Western blotting. The ERK1/2 level was also measured as an internal control. D–H. HUVECs were transfected as described above and were assessed for migration, tubulogenesis and proliferation after 48 h. D–E. Migration of transfected HUVECs in a scratch-wound assay 16 h after treatment with bFGF (10 ng/ml) and VEGFa (50 ng/ml) (n = 6–12 measurements/condition; n = 3 experiments). F–G. Transfected HUVECs were seeded onto Matrigel in EGM-2 and then allowed to form capillary-like structures for 16 h. Living cells were labeled with calcein-AM. Representative figures are shown in (F). Branching numbers (G) were quantified with Image J software (n = 5–10 pictures/condition; n = 3 experiments). H. Proliferation was assayed in transfected HUVEC treated with bFGF (10 ng/ml) and VEGFa (50 ng/ml) by measuring BrdU incorporation (n = 3). Data are means with the SD. *p<0.05 versus corresponding control.
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getmorefigures.php?uid=PMC3037403&req=5

pone-0016979-g005: RhoB-silencing alters endothelial cell migration, proliferation and tubulogenesis.A. Predicted miR-21 binding site within the RhoB 3′UTR. B. The wild-type (WT RhoB 3′UTR) or mutated (Mut RhoB 3′UTR) reporter plasmid was cotransfected into HEK293T cells with a precursor of miR-21 (Pre-21) or with a precursor control (Pre-Ctrl). Luciferase activities were quantified 48 h after transfection as described in the materials and methods section (Luciferase reporter assay and cloning). Renilla luciferase activity was normalized by Firefly luciferase activity. C. HUVECs were transfected with non-silencing siRNA (siRNA-Ctrl) or with RhoB siRNA and the RhoB protein level was measured after 48 h by Western blotting. The ERK1/2 level was also measured as an internal control. D–H. HUVECs were transfected as described above and were assessed for migration, tubulogenesis and proliferation after 48 h. D–E. Migration of transfected HUVECs in a scratch-wound assay 16 h after treatment with bFGF (10 ng/ml) and VEGFa (50 ng/ml) (n = 6–12 measurements/condition; n = 3 experiments). F–G. Transfected HUVECs were seeded onto Matrigel in EGM-2 and then allowed to form capillary-like structures for 16 h. Living cells were labeled with calcein-AM. Representative figures are shown in (F). Branching numbers (G) were quantified with Image J software (n = 5–10 pictures/condition; n = 3 experiments). H. Proliferation was assayed in transfected HUVEC treated with bFGF (10 ng/ml) and VEGFa (50 ng/ml) by measuring BrdU incorporation (n = 3). Data are means with the SD. *p<0.05 versus corresponding control.
Mentions: To confirm that RhoB is a direct target of miR-21, we constructed a luciferase reporter vector encoding the complete 3′UTR of RhoB (WT RhoB 3′UTR) as well as a control vector containing mismatches in predicted miR-21 binding site (Mut RhoB 3′UTR) (Figure 5A). Cotransfection of the WT RhoB 3′UTR plasmid and pre-miR-21 in HEK-293T cells resulted in a strong decrease in luciferase activity suggesting that RhoB mRNA is a direct target of miR-21. Importantly, mutations in the sequence targeted by miR-21 in RhoB 3′UTR reduced the observed down-regulation of luciferase activity by pre-miR-21 validating that this predicted binding site is necessary for miR-21 dependent RhoB expression (Figure 5B).

Bottom Line: Expression of miR-21 in endothelial cells also led to a reduction in the organization of actin into stress fibers, which may explain the decrease in cell migration.Further mechanistic studies showed that miR-21 targets RhoB, as revealed by a decrease in RhoB expression and activity in miR-21 overexpressing cells.Our results identify miR-21 as a new angiogenesis inhibitor and suggest that inhibition of cell migration and tubulogenesis is mediated through repression of RhoB.

View Article: PubMed Central - PubMed

Affiliation: Unit of Molecular Biology and Genetic Engineering, GIGA-Research, University of Liège, Sart Tilman, Liège, Belgium.

ABSTRACT

Background: MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that regulate gene expression at post-transcriptional level. The recent discovery of the involvement of these RNAs in the control of angiogenesis renders them very attractive in the development of new approaches for restoring the angiogenic balance. Whereas miRNA-21 has been demonstrated to be highly expressed in endothelial cells, the potential function of this miRNA in angiogenesis has never been investigated.

Methodology/principal findings: We first observed in endothelial cells a negative regulation of miR-21 expression by serum and bFGF, two pro-angiogenic factors. Then using in vitro angiogenic assays, we observed that miR-21 acts as a negative modulator of angiogenesis. miR-21 overexpression reduced endothelial cell proliferation, migration and the ability of these cells to form tubes whereas miR-21 inhibition using a LNA-anti-miR led to opposite effects. Expression of miR-21 in endothelial cells also led to a reduction in the organization of actin into stress fibers, which may explain the decrease in cell migration. Further mechanistic studies showed that miR-21 targets RhoB, as revealed by a decrease in RhoB expression and activity in miR-21 overexpressing cells. RhoB silencing impairs endothelial cell migration and tubulogenesis, thus providing a possible mechanism for miR-21 to inhibit angiogenesis. Finally, the therapeutic potential of miR-21 as an angiogenesis inhibitor was demonstrated in vivo in a mouse model of choroidal neovascularization.

Conclusions/significance: Our results identify miR-21 as a new angiogenesis inhibitor and suggest that inhibition of cell migration and tubulogenesis is mediated through repression of RhoB.

Show MeSH
Related in: MedlinePlus