When transfected with miR-200b, the power of HUVECs to create a capillary pipe about Matrigel and VEGF-induced phosphorylation of ERK1/2 were considerably reduced. Fig. 2. Down-regulation of VEGF, Flt-1, and KDR by miR-200b through immediate focusing on of 3-UTRs. (A) Traditional western blots of lysates from HUVECs transfected with miRNA mimics for 48 h. (B) At 48 h after transfection of HeLa cells with miRNA mimics, VEGF focus in the tradition medium was assessed by ELISA. Hypoxia was induced by DFX treatment for 24 h before harvesting the tradition medium. Results stand for the suggest SD from three 3rd party tests. *< 0.001, **< 0.01. (C) Schematic diagrams of binding sites for miR-200b in the 3- UTRs of VEGF, Flt-1, and KDR. The real number below the vertical bar indicates the nucleotide position from the binding site for miR-200b. (D) Direct focusing on of 3-UTRs of VEGF, Flt-1, and KDR by miR-200b as exposed by luciferase reporter assay. At 48 h after transfection with miRNA, luciferase activity was assessed in A549 cells. Normalized Renilla luciferase activity in cells transfected with NC was arranged at 100%. Luciferase activity of cells transfected with miR-200b and NC can be demonstrated as stuffed and open up pubs, respectively. Results stand for the suggest SD from three 3rd party tests. *< 0.001, **< 0.01, ***< 0.05. W, wild-type 3-UTR; M, mutated 3-UTR; 5M, mutated at 5 site in 3-UTR of KDR, 3M, mutated at 3 site in 3-UTR of KDR; DM, mutated at both 5 and 3 sites in 3-UTR of KDR. VEGF, Flt-1, and KDR are direct targets of miR-200b To validate VEGF, Flt-1, and KDR as targets of miR-200b, down-regulation of three genes at the protein level by miR-200b was examined by Western ELISA and blot analyses. Contained in these analyses was miR-200c Additionally, which shares the same seed sequence with miR-200b. As is seen in Fig. 2A, Flt-1 and KDR proteins in miR-200b- and miR-200ctransfected HUVECs were significantly decreased in comparison to NC-transfected cells. Although luciferase activity was significantly decreased when A549 cells were cotransfected with miR-24 and luciferase reporter plasmid harboring the 3-UTR of Flt-1 (Fig. 1), Western blot analysis revealed that Flt-1 protein had not been decreased after transfection of HUVECs with miR-24. Since VEGF is up-regulated and secreted under hypoxic conditions, miRNA-transfected HeLa cells were treated with DFX for 24 h to mimic hypoxia, and VEGF concentrations in culture supernatants were measured by ELISA. We observed how the VEGF level in miR-200b- and miR-200c-transfected HeLa cells was decreased to ~50% of this in NC-transfected cells under DFX-treated conditions (Fig. 2B). When transfected with miR- 20, which may target VEGF, secreted VEGF level was similar compared to that in miR-200b- and miR-200c-transfected cells (Hua et al., 2006). Together, these total results show that miR-200b and miR-200c down-regulate Flt-1, KDR, and VEGF in the protein level. To show direct interaction between miR-200b as well as the 3-UTRs of the 3 genes, we investigated the result of miR- 200b on the experience of 3-UTR-luciferase reporter plasmids. As is seen in Fig. 2D, cotransfection of miR-200b having a luciferase reporter plasmid bearing the 3-UTR of VEGF, Flt-1, or KDR led to significant decreases in luciferase activity in comparison to that in NC-transfected cells. Although luciferase activity after transfection with miR-200b was high in the case of VEGF somewhat, a similar degree of luciferase activity was seen in cells transfected with miR-20 (data not shown). To verify that miR-200b interacts using the predicted binding sites in the 3-UTRs, these websites were deleted from 3-UTR-luciferase reporter plasmids, as well as the resulting plasmids were cotransfected with miR-200b. We observed that luciferase activity had not been decreased when predicted binding sites were deleted through the 3-UTRs of VEGF, Flt-1, and KDR (Fig. 2D). In the full case of KDR, the two 2 binding sites in the 3-UTR synergistically seemed to function, using the downstream 3 binding site being far better. Furthermore to miR-200b, cotransfection with miR-200c yielded basically the same leads to luciferase reporter assays using wild-type and mutant 3-UTR-luciferase reporter plasmids (data not shown). Taken together, these data indicate that miR-200b regulates VEGF directly, Flt-1, and KDR through interaction with predicted binding sites within their 3-UTRs. Tube formation is inhibited by miR-200b/-200c Since.Intriguingly, both VEGF and its own receptors (Flt-1 and KDR) were negatively regulated by miR-200b. 200b negatively regulates VEGF signaling by targeting VEGF a nd i ts r eceptors a nd t hat miR- 200b may have therapeutic potential as an angiogenesis inhibitor. < 0.01, ***< 0.05. Open in another window Fig. 2. Down-regulation of VEGF, Flt-1, and KDR by miR-200b through direct targeting of 3-UTRs. (A) Western blots of lysates from HUVECs transfected with miRNA mimics for 48 h. (B) At 48 h after transfection of HeLa cells with miRNA mimics, VEGF concentration in the culture medium was measured by ELISA. Hypoxia was induced by DFX treatment for 24 h before harvesting the culture medium. Results represent the mean SD from three independent experiments. *< 0.001, **< 0.01. (C) Schematic diagrams of binding sites for miR-200b in the 3- UTRs of VEGF, Flt-1, and KDR. The quantity below the vertical bar indicates the nucleotide position from the binding site for miR-200b. (D) Direct targeting of 3-UTRs of VEGF, Flt-1, and KDR by miR-200b as revealed by luciferase reporter assay. At 48 h after transfection with miRNA, luciferase activity was measured in A549 cells. Normalized Renilla luciferase activity in cells transfected with NC was set at 100%. Luciferase activity of cells transfected with NC and miR-200b is shown as filled and open bars, respectively. Results represent the mean SD from three independent experiments. *< 0.001, **< 0.01, ***< 0.05. W, wild-type 3-UTR; M, mutated 3-UTR; 5M, mutated at 5 site in 3-UTR of KDR, 3M, mutated at 3 site in 3-UTR of KDR; DM, mutated at both 5 and 3 sites in 3-UTR of KDR. VEGF, Flt-1, and KDR are direct targets of miR-200b To validate VEGF, Flt-1, and KDR as targets of miR-200b, down-regulation of three genes in the protein level by miR-200b was examined by Western blot and ELISA analyses. Additionally contained in these analyses was miR-200c, which shares the same seed sequence with miR-200b. As is seen in Fig. 2A, Flt-1 and KDR proteins in miR-200b- and miR-200ctransfected HUVECs were significantly decreased in comparison to NC-transfected cells. Although luciferase activity was significantly decreased when A549 cells were cotransfected with miR-24 and luciferase reporter plasmid harboring the 3-UTR of Flt-1 (Fig. 1), Western blot analysis revealed that Flt-1 protein had not been decreased after transfection of HUVECs with miR-24. Since VEGF is up-regulated and secreted under hypoxic conditions, miRNA-transfected HeLa cells were treated with DFX for 24 h to mimic hypoxia, and VEGF concentrations in culture supernatants were measured by ELISA. We observed how the VEGF level in miR-200b- and miR-200c-transfected HeLa cells was decreased to ~50% of this in NC-transfected cells under DFX-treated conditions (Fig. 2B). When transfected with miR- 20, which may target VEGF, secreted VEGF level was similar compared to that in miR-200b- and miR-200c-transfected cells (Hua et al., 2006). Together, these results show that miR-200b and miR-200c down-regulate Flt-1, KDR, and VEGF in the protein level. To show direct interaction between miR-200b as well as the 3-UTRs of the 3 genes, we investigated the result of miR- 200b on the experience of 3-UTR-luciferase reporter plasmids. As is seen in Fig. 2D, cotransfection of miR-200b having a luciferase reporter plasmid bearing the 3-UTR of VEGF, Flt-1, or KDR led to significant decreases in luciferase activity in comparison to that in NC-transfected cells. Although luciferase activity after transfection with miR-200b was somewhat saturated in the situation of VEGF, an identical degree of luciferase activity was seen in cells transfected with miR-20 (data not shown). To verify that miR-200b interacts using the predicted binding sites in the 3-UTRs, these websites were deleted from 3-UTR-luciferase reporter plasmids, as well as the resulting plasmids were cotransfected with miR-200b. We observed that luciferase activity had not been decreased when predicted binding sites were deleted through the 3-UTRs of VEGF, Flt-1, and KDR (Fig. 2D). Regarding KDR, the two 2 binding sites in the 3-UTR seemed to function synergistically, using the downstream 3 binding site being far better. Furthermore to miR-200b, cotransfection with miR-200c yielded basically the same leads to luciferase reporter assays using wild-type and mutant 3-UTR-luciferase reporter plasmids (data not shown). Taken together, these data indicate that miR-200b directly regulates VEGF, Flt-1, and KDR through interaction with predicted binding sites within their 3-UTRs. Tube formation is inhibited by miR-200b/-200c Since capillary tube formation on Matrigel can be an essential angiogenic property of HUVECs, we investigated whether downregulation of Flt-1 and KDR by miR-200b and miR-200c affects pipe formation. At 48 h after transfection, HUVECs were serum- starved overnight and the next day, HUVECs were cultured on the Matrigel-coated 12-well plate for 6 h. As is seen in Fig. 3,.In A549 cells, miR-200b targeted the predicted binding sites in the 3-untranslated region (3-UTR) of VEGF, Flt-1, and KDR as revealed with a luciferase reporter assay. ***< 0.05. Open in another window Fig. 2. Down-regulation of VEGF, Flt-1, and KDR by miR-200b through direct targeting of 3-UTRs. (A) Western blots of lysates from HUVECs transfected with miRNA mimics for 48 h. (B) At 48 h after transfection of HeLa cells with miRNA mimics, VEGF concentration in the culture medium was measured by ELISA. Hypoxia was induced by DFX treatment for 24 h before harvesting the culture medium. Results represent the mean SD from three independent experiments. *< 0.001, **< 0.01. (C) Schematic diagrams of binding sites for miR-200b in the 3- UTRs of VEGF, Flt-1, and KDR. The quantity below the vertical bar indicates the nucleotide position from the binding site for miR-200b. (D) Direct targeting of 3-UTRs of VEGF, Flt-1, and KDR by miR-200b as revealed by luciferase reporter assay. At 48 h after transfection with miRNA, luciferase activity was measured in A549 cells. Normalized Renilla luciferase activity in cells transfected with NC was set at 100%. Luciferase activity of cells transfected with miR-200b and NC can be demonstrated as stuffed and open up pubs, respectively. Results represent the mean SD from three independent experiments. *< 0.001, **< 0.01, ***< 0.05. W, wild-type 3-UTR; M, mutated 3-UTR; 5M, mutated at 5 site in 3-UTR of KDR, 3M, mutated at 3 site in 3-UTR of KDR; DM, mutated at both 5 and 3 sites in 3-UTR of KDR. VEGF, Flt-1, and KDR are direct targets of miR-200b To validate VEGF, Flt-1, and KDR as targets of miR-200b, down-regulation of three genes in the protein level by miR-200b was examined by Western blot and ELISA analyses. Additionally contained in these analyses was miR-200c, which shares the same seed sequence with miR-200b. As is seen in Fig. 2A, AG-120 Flt-1 and AG-120 KDR proteins in miR-200b- and miR-200ctransfected HUVECs were significantly decreased in comparison to NC-transfected cells. Although luciferase activity was significantly decreased when A549 cells were cotransfected with miR-24 and luciferase reporter plasmid harboring the 3-UTR of Flt-1 (Fig. 1), Western blot analysis revealed that Flt-1 protein had not been decreased after transfection of HUVECs with miR-24. Since VEGF is up-regulated and secreted under hypoxic conditions, miRNA-transfected HeLa cells were treated with DFX for 24 h to mimic hypoxia, and VEGF concentrations in culture supernatants were measured by ELISA. We observed how the VEGF level in miR-200b- and miR-200c-transfected HeLa cells was decreased to ~50% of this in NC-transfected cells under DFX-treated conditions (Fig. 2B). When transfected with miR- 20, which may target VEGF, secreted VEGF level was similar compared to that in miR-200b- and miR-200c-transfected cells (Hua et al., 2006). Together, these results show that miR-200b and miR-200c down-regulate Flt-1, KDR, and VEGF in the protein level. To show direct interaction between miR-200b as well as the 3-UTRs of the 3 genes, we investigated the result of miR- 200b on the experience of 3-UTR-luciferase reporter plasmids. As is seen in Fig. 2D, cotransfection of miR-200b having a luciferase reporter plasmid bearing the 3-UTR of VEGF, Flt-1, or KDR led to significant decreases in luciferase activity in comparison to that in NC-transfected cells. Although luciferase activity after transfection with miR-200b was somewhat saturated in the situation of VEGF, an identical degree of luciferase activity was seen in cells transfected with miR-20 (data not shown). To verify that miR-200b interacts using the predicted binding sites in the 3-UTRs, these websites were deleted from 3-UTR-luciferase reporter plasmids, as well as the resulting plasmids were cotransfected with miR-200b. We observed that luciferase activity had not been decreased when predicted binding sites were deleted through the 3-UTRs of VEGF, Flt-1, and KDR (Fig. 2D). Regarding KDR, the two 2 binding sites in the 3-UTR seemed to function synergistically, using the downstream 3 binding site being far better. Furthermore to miR-200b,.Taking into consideration the inhibitory aftereffect of miR-200 on VEGF signaling, chances are how the miR-200 family members is expressed or silenced in low amounts in HUVECs. Two recent documents reported that miR-200b is involved with angiogenesis. separate home window Fig. 2. Down-regulation of VEGF, Flt-1, and KDR by miR-200b through immediate focusing on of 3-UTRs. (A) Traditional western blots of lysates from HUVECs transfected with miRNA mimics for 48 h. (B) At 48 h after transfection of HeLa cells with miRNA mimics, VEGF focus in the tradition medium was assessed by ELISA. Hypoxia was induced by DFX treatment for 24 h before harvesting the tradition medium. Results stand for the suggest SD from three 3rd party tests. *< 0.001, **< 0.01. (C) Schematic diagrams of binding sites for miR-200b in the 3- UTRs of VEGF, Flt-1, and KDR. The quantity below the vertical pub shows the nucleotide placement from the binding site for miR-200b. (D) Direct targeting of 3-UTRs of VEGF, Flt-1, and KDR by miR-200b as revealed by luciferase reporter assay. At 48 h after transfection with miRNA, luciferase activity was measured in A549 cells. Normalized Renilla luciferase activity in cells transfected with NC was set at 100%. Luciferase activity of cells transfected with NC and miR-200b is shown as filled and open bars, respectively. Results represent the mean SD from three independent experiments. *< 0.001, **< 0.01, ***< 0.05. W, wild-type 3-UTR; M, mutated 3-UTR; 5M, mutated at 5 site in 3-UTR of KDR, 3M, mutated at 3 site in 3-UTR of KDR; DM, mutated at both 5 and 3 sites in 3-UTR of KDR. VEGF, Flt-1, and KDR are direct targets of miR-200b To validate VEGF, Flt-1, and KDR as targets of miR-200b, down-regulation of three genes at the protein level by miR-200b was examined by Western blot and ELISA analyses. Additionally included in these analyses was miR-200c, which shares the same seed sequence with miR-200b. As can be seen in Fig. 2A, Flt-1 and KDR proteins in miR-200b- and miR-200ctransfected HUVECs were significantly decreased compared to NC-transfected cells. Although luciferase activity PRKM1 was significantly decreased when A549 cells were cotransfected with miR-24 and luciferase reporter plasmid harboring the 3-UTR of Flt-1 (Fig. 1), Western blot analysis revealed that Flt-1 protein was not decreased after transfection of HUVECs with miR-24. Since VEGF is up-regulated and secreted under hypoxic conditions, miRNA-transfected HeLa cells were treated with DFX for 24 h to mimic hypoxia, and VEGF concentrations in culture supernatants were measured by ELISA. We observed that the VEGF level in miR-200b- and miR-200c-transfected HeLa cells was decreased to ~50% of that in NC-transfected cells under DFX-treated conditions (Fig. 2B). When transfected with miR- 20, which is known to target VEGF, secreted VEGF level was similar to that in miR-200b- and miR-200c-transfected cells (Hua et al., 2006). Together, these results show that miR-200b and miR-200c down-regulate Flt-1, KDR, and VEGF at the protein level. To demonstrate direct interaction between miR-200b and the 3-UTRs of these 3 genes, we investigated the effect of miR- 200b on the activity of 3-UTR-luciferase reporter plasmids. As can be seen in Fig. 2D, cotransfection of miR-200b with a luciferase reporter plasmid bearing the 3-UTR of VEGF, Flt-1, or KDR resulted in significant decreases in luciferase activity compared to that in NC-transfected cells. Although luciferase activity after transfection with miR-200b was somewhat high in the case of VEGF, a similar level of luciferase activity was observed in cells transfected with miR-20 (data not shown). To verify that miR-200b interacts with the predicted binding sites in the 3-UTRs, these sites were deleted from 3-UTR-luciferase reporter plasmids, and the resulting plasmids were cotransfected with miR-200b. We observed that luciferase activity was not decreased when predicted binding sites were deleted from the 3-UTRs of VEGF, Flt-1, and KDR (Fig. 2D). In the case of KDR, the 2 2 binding sites in the 3-UTR appeared to function synergistically, with the downstream 3 binding site being more effective. In addition to miR-200b, cotransfection with miR-200c yielded essentially the same results in luciferase reporter.Luciferase activity of cells transfected with NC and miR-200b is shown as filled and open bars, respectively. in a separate window Fig. 2. Down-regulation of VEGF, Flt-1, and KDR by miR-200b through direct targeting of 3-UTRs. (A) Western blots of lysates from HUVECs transfected with miRNA mimics for 48 h. (B) At 48 h after transfection of HeLa cells with miRNA mimics, VEGF concentration in the culture medium was measured by ELISA. Hypoxia was induced by DFX treatment for 24 h before harvesting the culture medium. Results represent the mean SD from three independent experiments. *< 0.001, **< 0.01. (C) Schematic diagrams of binding sites for miR-200b in the 3- UTRs of VEGF, Flt-1, and KDR. The number below the vertical bar indicates the nucleotide position of the binding site for miR-200b. (D) Direct targeting of 3-UTRs of VEGF, Flt-1, and KDR by miR-200b as revealed by luciferase reporter assay. At 48 h after transfection with miRNA, luciferase activity was measured in A549 cells. Normalized Renilla luciferase activity in cells transfected with NC was set at 100%. Luciferase activity of cells transfected with NC and miR-200b is shown as filled and open bars, respectively. Results represent the mean SD from three independent experiments. *< 0.001, **< 0.01, ***< 0.05. W, wild-type 3-UTR; M, mutated 3-UTR; 5M, mutated at 5 site in 3-UTR of KDR, 3M, mutated at 3 site in 3-UTR of KDR; DM, mutated at both 5 and 3 sites in 3-UTR of KDR. VEGF, Flt-1, and KDR are direct targets of miR-200b To validate VEGF, Flt-1, and KDR as targets of miR-200b, down-regulation of three genes at the protein level by miR-200b was examined by Western blot and ELISA analyses. Additionally included in these analyses was miR-200c, which shares the same seed sequence with miR-200b. As can be seen in Fig. 2A, Flt-1 and KDR proteins in miR-200b- and miR-200ctransfected HUVECs were significantly decreased compared to NC-transfected cells. Although luciferase activity was significantly decreased when A549 cells were cotransfected with miR-24 and luciferase reporter plasmid harboring the 3-UTR of Flt-1 (Fig. 1), Western blot analysis revealed that Flt-1 protein was not decreased after transfection of HUVECs with miR-24. Since VEGF is up-regulated and secreted under hypoxic conditions, miRNA-transfected HeLa cells were treated with DFX for 24 h to mimic hypoxia, and VEGF concentrations in culture supernatants were measured by ELISA. We observed that the VEGF level in miR-200b- and miR-200c-transfected HeLa cells was decreased to ~50% of that in NC-transfected cells under DFX-treated conditions (Fig. 2B). When transfected with miR- 20, which is known to target VEGF, secreted VEGF level was similar to that in miR-200b- and miR-200c-transfected cells (Hua et al., 2006). Together, these results show that miR-200b and miR-200c down-regulate Flt-1, KDR, and VEGF at the protein level. To demonstrate direct interaction between miR-200b and the 3-UTRs of these 3 genes, we investigated the effect of miR- AG-120 200b on the activity of 3-UTR-luciferase reporter plasmids. As can be seen in Fig. 2D, cotransfection of miR-200b with a luciferase reporter plasmid bearing the 3-UTR of VEGF, Flt-1, or KDR resulted in significant decreases in luciferase activity compared to that in NC-transfected cells. Although luciferase activity after transfection with miR-200b was somewhat high in the case of VEGF, a similar level of luciferase activity was observed in cells transfected with miR-20 (data not shown). To verify that miR-200b interacts with the predicted binding sites in the 3-UTRs, these sites were deleted from 3-UTR-luciferase reporter plasmids, and the resulting plasmids were cotransfected with miR-200b. We observed that luciferase activity was not decreased when predicted binding sites were deleted from the 3-UTRs of VEGF, Flt-1, and KDR (Fig. 2D). In the case of KDR, the 2 2 binding sites in the 3-UTR appeared to function synergistically, with the downstream 3 binding site being more effective. In addition to miR-200b, cotransfection with miR-200c yielded essentially the same results in luciferase reporter assays using wild-type and mutant 3-UTR-luciferase reporter plasmids (data not shown). Taken together, these data indicate that miR-200b directly regulates VEGF, Flt-1, and KDR through interaction with predicted binding sites in their 3-UTRs. Tube formation is inhibited by miR-200b/-200c Since capillary tube formation on Matrigel is an essential angiogenic property of HUVECs, we investigated whether downregulation of KDR and Flt-1 by miR-200b and miR-200c affects tube formation. At 48 h after transfection, HUVECs were serum- starved overnight and the following day, HUVECs were cultured on a Matrigel-coated 12-well plate for 6 h. As can be seen in Fig. 3, NC-transfected HUVECs formed well-organized capillary-like structures. However, transfection.