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Overexpression of miRNA-497 inhibits tumor angiogenesis by targeting VEGFR2.

Tu Y, Liu L, Zhao D, Liu Y, Ma X, Fan Y, Wan L, Huang T, Cheng Z, Shen B - Sci Rep (2015)

Bottom Line: The results demonstrated that overexpression of miR-497 showed inhibitory effects on VEGFR2 activation and downstream Raf/MEK/ERK signal pathways in vitro and in vivo.Moreover, overexpression of miR-497 effectively induced HUVECs apoptosis by targeting VEGFR2 and downstream PI3K/AKT signaling pathway.Furthermore, miR-497 exhibited anti-angiogenesis and anti-tumor effects in the VEGFR2-luc breast tumor model proven by BLI, WB and immunohistochemistry analysis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular Imaging, College of Heilongjiang Province, Harbin, Heilongjiang, China.

ABSTRACT
Recent studies reported miR-497 exhibited inhibitory effects in various cancers. However, whether miR-497 is involved in inhibiting angiogenesis, which is critical for tumor growth and metastasis, is still unknown. The purpose of this study was to investigate the potential role of miR-497 in tumor angiogenesis. In this work, cell proliferation and apoptosis analyses were conducted to explore the potential function of miR-497 in HUVECs by using MTT and TUNEL assays. Western blotting (WB) was employed to validate the downstream targets of miR-497. Furthermore, in order to disclose the role of miR-497 on angiogenesis, VEGFR2-luc transgenic mice were treated with miR-497 mimic and applied to monitor tumor angiogenesis and growth by in vivo bioluminescent imaging (BLI). The results demonstrated that overexpression of miR-497 showed inhibitory effects on VEGFR2 activation and downstream Raf/MEK/ERK signal pathways in vitro and in vivo. Moreover, overexpression of miR-497 effectively induced HUVECs apoptosis by targeting VEGFR2 and downstream PI3K/AKT signaling pathway. Furthermore, miR-497 exhibited anti-angiogenesis and anti-tumor effects in the VEGFR2-luc breast tumor model proven by BLI, WB and immunohistochemistry analysis. In summary, miR-497 inhibits tumor angiogenesis and growth via targeting VEGFR2, indicating miR-497 can be explored as a potential drug candidate for cancer therapy.

No MeSH data available.


Related in: MedlinePlus

Up-regulation of miR-497 mitigated VEGFR2 level and increased the levels of apoptosis related proteins in human umbilical vein endothelial cells (HUVECs).(A) Using qRT-PCR analysis, the expression of miR-497 was increased in miR-497 mimic treatment and decreased in miR-497 inhibitor treatment in HUVECs. Data were expressed as mean ± SEM, n = 3; **P < 0.01 vs. control group, ##P < 0.01 vs. control group. (B–F) Protein levels of VEGFR2, Total Akt, p-Akt, Bax, and Bcl-2 were detected by western blotting assay. Data were expressed as mean ± SEM, n = 3; **P < 0.01 or ***P < 0.001 vs. control group.
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f1: Up-regulation of miR-497 mitigated VEGFR2 level and increased the levels of apoptosis related proteins in human umbilical vein endothelial cells (HUVECs).(A) Using qRT-PCR analysis, the expression of miR-497 was increased in miR-497 mimic treatment and decreased in miR-497 inhibitor treatment in HUVECs. Data were expressed as mean ± SEM, n = 3; **P < 0.01 vs. control group, ##P < 0.01 vs. control group. (B–F) Protein levels of VEGFR2, Total Akt, p-Akt, Bax, and Bcl-2 were detected by western blotting assay. Data were expressed as mean ± SEM, n = 3; **P < 0.01 or ***P < 0.001 vs. control group.

Mentions: One of the predicted targets of miR-497 is VEGFR2 by using the specific program TargetScan (http://www.targetscan.org) (see Supplementary material online, Fig. S1A). In order to further demonstrate that miR-497 may inhibit VEGFR2 expression through direct interaction with predicted binding sites located in the 3′-UTR region of VEGFR2 in HUVECs, we cloned a reporter vector in which luciferase cDNA was followed by a fragment of the 3′-UTR from VEGFR2 mRNA containing the putative miR-497 binding sequences. Furthermore, we synthesized another luciferase reporter fused to the VEGFR2 3′-UTRs, but with a mutant miR-497 binding sequence. We then transfected this luciferase reporter vector with either wild-type or mutant miR-497 binding sequences into HUVECs. We also co-transfected these cells with miR-497 mimic or mimic control and measured luciferase activity. As shown in Fig. 1B, the miR-497 mimic administered at a concentration of 50 nM decreases luciferase activity of the reporter vector containing miR-497 binding sequences (see Supplementary material online, Fig. S1B). However, the miR-497 mimic has no significant effect on the reporter vector with mutated miR-497 binding sequences (see Supplementary material online, Fig. S1C). These data suggest that miR-497 may inhibit translation of VEGFR2 by directly acting on response elements specific for miR-497 in the VEGFR2 3′-UTR region in HUVECs.


Overexpression of miRNA-497 inhibits tumor angiogenesis by targeting VEGFR2.

Tu Y, Liu L, Zhao D, Liu Y, Ma X, Fan Y, Wan L, Huang T, Cheng Z, Shen B - Sci Rep (2015)

Up-regulation of miR-497 mitigated VEGFR2 level and increased the levels of apoptosis related proteins in human umbilical vein endothelial cells (HUVECs).(A) Using qRT-PCR analysis, the expression of miR-497 was increased in miR-497 mimic treatment and decreased in miR-497 inhibitor treatment in HUVECs. Data were expressed as mean ± SEM, n = 3; **P < 0.01 vs. control group, ##P < 0.01 vs. control group. (B–F) Protein levels of VEGFR2, Total Akt, p-Akt, Bax, and Bcl-2 were detected by western blotting assay. Data were expressed as mean ± SEM, n = 3; **P < 0.01 or ***P < 0.001 vs. control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561885&req=5

f1: Up-regulation of miR-497 mitigated VEGFR2 level and increased the levels of apoptosis related proteins in human umbilical vein endothelial cells (HUVECs).(A) Using qRT-PCR analysis, the expression of miR-497 was increased in miR-497 mimic treatment and decreased in miR-497 inhibitor treatment in HUVECs. Data were expressed as mean ± SEM, n = 3; **P < 0.01 vs. control group, ##P < 0.01 vs. control group. (B–F) Protein levels of VEGFR2, Total Akt, p-Akt, Bax, and Bcl-2 were detected by western blotting assay. Data were expressed as mean ± SEM, n = 3; **P < 0.01 or ***P < 0.001 vs. control group.
Mentions: One of the predicted targets of miR-497 is VEGFR2 by using the specific program TargetScan (http://www.targetscan.org) (see Supplementary material online, Fig. S1A). In order to further demonstrate that miR-497 may inhibit VEGFR2 expression through direct interaction with predicted binding sites located in the 3′-UTR region of VEGFR2 in HUVECs, we cloned a reporter vector in which luciferase cDNA was followed by a fragment of the 3′-UTR from VEGFR2 mRNA containing the putative miR-497 binding sequences. Furthermore, we synthesized another luciferase reporter fused to the VEGFR2 3′-UTRs, but with a mutant miR-497 binding sequence. We then transfected this luciferase reporter vector with either wild-type or mutant miR-497 binding sequences into HUVECs. We also co-transfected these cells with miR-497 mimic or mimic control and measured luciferase activity. As shown in Fig. 1B, the miR-497 mimic administered at a concentration of 50 nM decreases luciferase activity of the reporter vector containing miR-497 binding sequences (see Supplementary material online, Fig. S1B). However, the miR-497 mimic has no significant effect on the reporter vector with mutated miR-497 binding sequences (see Supplementary material online, Fig. S1C). These data suggest that miR-497 may inhibit translation of VEGFR2 by directly acting on response elements specific for miR-497 in the VEGFR2 3′-UTR region in HUVECs.

Bottom Line: The results demonstrated that overexpression of miR-497 showed inhibitory effects on VEGFR2 activation and downstream Raf/MEK/ERK signal pathways in vitro and in vivo.Moreover, overexpression of miR-497 effectively induced HUVECs apoptosis by targeting VEGFR2 and downstream PI3K/AKT signaling pathway.Furthermore, miR-497 exhibited anti-angiogenesis and anti-tumor effects in the VEGFR2-luc breast tumor model proven by BLI, WB and immunohistochemistry analysis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular Imaging, College of Heilongjiang Province, Harbin, Heilongjiang, China.

ABSTRACT
Recent studies reported miR-497 exhibited inhibitory effects in various cancers. However, whether miR-497 is involved in inhibiting angiogenesis, which is critical for tumor growth and metastasis, is still unknown. The purpose of this study was to investigate the potential role of miR-497 in tumor angiogenesis. In this work, cell proliferation and apoptosis analyses were conducted to explore the potential function of miR-497 in HUVECs by using MTT and TUNEL assays. Western blotting (WB) was employed to validate the downstream targets of miR-497. Furthermore, in order to disclose the role of miR-497 on angiogenesis, VEGFR2-luc transgenic mice were treated with miR-497 mimic and applied to monitor tumor angiogenesis and growth by in vivo bioluminescent imaging (BLI). The results demonstrated that overexpression of miR-497 showed inhibitory effects on VEGFR2 activation and downstream Raf/MEK/ERK signal pathways in vitro and in vivo. Moreover, overexpression of miR-497 effectively induced HUVECs apoptosis by targeting VEGFR2 and downstream PI3K/AKT signaling pathway. Furthermore, miR-497 exhibited anti-angiogenesis and anti-tumor effects in the VEGFR2-luc breast tumor model proven by BLI, WB and immunohistochemistry analysis. In summary, miR-497 inhibits tumor angiogenesis and growth via targeting VEGFR2, indicating miR-497 can be explored as a potential drug candidate for cancer therapy.

No MeSH data available.


Related in: MedlinePlus