Limits...
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.

Show MeSH

Related in: MedlinePlus

miR-21 reduces RhoB expression and activity in endothelial cells.A. Alignment of potential miR-21 binding sites in the 3′UTR of the RhoB mRNA of different species. B. HUVECs were transfected with a precursor of miR-21 (Pre-miR-21) or with a precursor control (Pre-miR-Ctrl) and with a LNA-21 or with a LNA control (LNA Ctrl). The RhoB mRNA level was analyzed after 48 h by qRT-PCR. C–D. Total protein was extracted from HUVECs 48 h post transfection and RhoA, RhoB and RhoC protein levels were measured by Western blotting. ERK1/2 level was analyzed as an internal control. D. Quantification of (C). Quantification was performed using ImageJ software. E–F. Measurement of GTPase activity of RhoB. E. Transfected HUVECs were processed for pull-down assays and Western blot analysis with specific antibody to RhoB. F. Quantification of (E). Quantification was performed using ImageJ software. G. Transfected HUVECs were seeded onto gelatin-coated slides and analyzed for fluorescence labeling by phalloidin-FITC (green). Nuclei were visualized by DAPI staining (blue). White arrows show areas with less actin stress fibers. Asterisks represent some elongated HUVECs. Arrowhead indicates stress fibers in the center of the cell. Pictures are representative of three independent experiments. Data are means with the SD. *p<0.05 versus corresponding control; (n = 3).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3037403&req=5

pone-0016979-g004: miR-21 reduces RhoB expression and activity in endothelial cells.A. Alignment of potential miR-21 binding sites in the 3′UTR of the RhoB mRNA of different species. B. HUVECs were transfected with a precursor of miR-21 (Pre-miR-21) or with a precursor control (Pre-miR-Ctrl) and with a LNA-21 or with a LNA control (LNA Ctrl). The RhoB mRNA level was analyzed after 48 h by qRT-PCR. C–D. Total protein was extracted from HUVECs 48 h post transfection and RhoA, RhoB and RhoC protein levels were measured by Western blotting. ERK1/2 level was analyzed as an internal control. D. Quantification of (C). Quantification was performed using ImageJ software. E–F. Measurement of GTPase activity of RhoB. E. Transfected HUVECs were processed for pull-down assays and Western blot analysis with specific antibody to RhoB. F. Quantification of (E). Quantification was performed using ImageJ software. G. Transfected HUVECs were seeded onto gelatin-coated slides and analyzed for fluorescence labeling by phalloidin-FITC (green). Nuclei were visualized by DAPI staining (blue). White arrows show areas with less actin stress fibers. Asterisks represent some elongated HUVECs. Arrowhead indicates stress fibers in the center of the cell. Pictures are representative of three independent experiments. Data are means with the SD. *p<0.05 versus corresponding control; (n = 3).

Mentions: In order to identify putative target genes regulated by miR-21 and involved in the control of angiogenesis, we evaluated targets computationally predicted by publicly available algorithms (Targetscan). Using these lists of in silico predicted targets, we searched for genes that may play a role in angiogenic processes. Among them we found genes encoding several regulators of endothelial cell functions and vessel growth, such as the RhoGTPase RhoB, Sox7, the transforming growth factor beta receptor II (TGFBRII) and the regulator of ERK activation Sprouty1 (SPRY1). Regulators of cell migration such as the Rho guanine nucleotide exchange factor 12 (ARHGEF12), the myosin phosphatase Rho interacting protein (MPRIP) and vinculin (VCL) are also listed as putative targets of miR-21. Analysis performed by qRT-PCR and/or by Western blotting revealed that SOX7, TGFBRII, ARHGEF12, MPRIP and VCL expression was unaffected by miR-21 overexpression in HUVECs while SPRY1 was regulated by miR-21 as revealed by Western blot (Figure S3A-B). Interestingly, the 3′UTR of the RhoB mRNA was found to contain one predicted binding site for miR-21 conserved between several species (Figure 4A), which suggest that RhoB might be a direct target of miR-21. Moreover, RhoB was reported very recently to be targeted by miR-21 in hepatocellular carcinoma cell lines. In HUVECs, overexpression of miR-21 significantly decreased the level of RhoB mRNA expression (Figure 4B). By contrast, inhibition of miR-21 expression increased RhoB mRNA. Regulation of endogenous RhoB expression by pre-miR-21 and LNA-21 was further confirmed at the protein level by Western blotting (Figures 4C-D). RhoB belongs to a family of small GTPases composed of three closely related homologs, A, B and C [28]. In addition, we demonstrated that the regulation of RhoB mediated by miR-21 was specific for this GTPase, since induction or repression of miR-21 had no significant effect on the expression of the other two related RhoGTPases, RhoA and RhoC (Figure 4C-D).


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)

miR-21 reduces RhoB expression and activity in endothelial cells.A. Alignment of potential miR-21 binding sites in the 3′UTR of the RhoB mRNA of different species. B. HUVECs were transfected with a precursor of miR-21 (Pre-miR-21) or with a precursor control (Pre-miR-Ctrl) and with a LNA-21 or with a LNA control (LNA Ctrl). The RhoB mRNA level was analyzed after 48 h by qRT-PCR. C–D. Total protein was extracted from HUVECs 48 h post transfection and RhoA, RhoB and RhoC protein levels were measured by Western blotting. ERK1/2 level was analyzed as an internal control. D. Quantification of (C). Quantification was performed using ImageJ software. E–F. Measurement of GTPase activity of RhoB. E. Transfected HUVECs were processed for pull-down assays and Western blot analysis with specific antibody to RhoB. F. Quantification of (E). Quantification was performed using ImageJ software. G. Transfected HUVECs were seeded onto gelatin-coated slides and analyzed for fluorescence labeling by phalloidin-FITC (green). Nuclei were visualized by DAPI staining (blue). White arrows show areas with less actin stress fibers. Asterisks represent some elongated HUVECs. Arrowhead indicates stress fibers in the center of the cell. Pictures are representative of three independent experiments. Data are means with the SD. *p<0.05 versus corresponding control; (n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037403&req=5

pone-0016979-g004: miR-21 reduces RhoB expression and activity in endothelial cells.A. Alignment of potential miR-21 binding sites in the 3′UTR of the RhoB mRNA of different species. B. HUVECs were transfected with a precursor of miR-21 (Pre-miR-21) or with a precursor control (Pre-miR-Ctrl) and with a LNA-21 or with a LNA control (LNA Ctrl). The RhoB mRNA level was analyzed after 48 h by qRT-PCR. C–D. Total protein was extracted from HUVECs 48 h post transfection and RhoA, RhoB and RhoC protein levels were measured by Western blotting. ERK1/2 level was analyzed as an internal control. D. Quantification of (C). Quantification was performed using ImageJ software. E–F. Measurement of GTPase activity of RhoB. E. Transfected HUVECs were processed for pull-down assays and Western blot analysis with specific antibody to RhoB. F. Quantification of (E). Quantification was performed using ImageJ software. G. Transfected HUVECs were seeded onto gelatin-coated slides and analyzed for fluorescence labeling by phalloidin-FITC (green). Nuclei were visualized by DAPI staining (blue). White arrows show areas with less actin stress fibers. Asterisks represent some elongated HUVECs. Arrowhead indicates stress fibers in the center of the cell. Pictures are representative of three independent experiments. Data are means with the SD. *p<0.05 versus corresponding control; (n = 3).
Mentions: In order to identify putative target genes regulated by miR-21 and involved in the control of angiogenesis, we evaluated targets computationally predicted by publicly available algorithms (Targetscan). Using these lists of in silico predicted targets, we searched for genes that may play a role in angiogenic processes. Among them we found genes encoding several regulators of endothelial cell functions and vessel growth, such as the RhoGTPase RhoB, Sox7, the transforming growth factor beta receptor II (TGFBRII) and the regulator of ERK activation Sprouty1 (SPRY1). Regulators of cell migration such as the Rho guanine nucleotide exchange factor 12 (ARHGEF12), the myosin phosphatase Rho interacting protein (MPRIP) and vinculin (VCL) are also listed as putative targets of miR-21. Analysis performed by qRT-PCR and/or by Western blotting revealed that SOX7, TGFBRII, ARHGEF12, MPRIP and VCL expression was unaffected by miR-21 overexpression in HUVECs while SPRY1 was regulated by miR-21 as revealed by Western blot (Figure S3A-B). Interestingly, the 3′UTR of the RhoB mRNA was found to contain one predicted binding site for miR-21 conserved between several species (Figure 4A), which suggest that RhoB might be a direct target of miR-21. Moreover, RhoB was reported very recently to be targeted by miR-21 in hepatocellular carcinoma cell lines. In HUVECs, overexpression of miR-21 significantly decreased the level of RhoB mRNA expression (Figure 4B). By contrast, inhibition of miR-21 expression increased RhoB mRNA. Regulation of endogenous RhoB expression by pre-miR-21 and LNA-21 was further confirmed at the protein level by Western blotting (Figures 4C-D). RhoB belongs to a family of small GTPases composed of three closely related homologs, A, B and C [28]. In addition, we demonstrated that the regulation of RhoB mediated by miR-21 was specific for this GTPase, since induction or repression of miR-21 had no significant effect on the expression of the other two related RhoGTPases, RhoA and RhoC (Figure 4C-D).

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