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Suppression of cell growth and invasion by miR-205 in breast cancer.

Wu H, Zhu S, Mo YY - Cell Res. (2009)

Bottom Line: In this study, we report that miR-205 is significantly underexpressed in breast tumor compared to the matched normal breast tissue.Of interest, ectopic expression of miR-205 significantly inhibits cell proliferation and anchorage independent growth, as well as cell invasion.Furthermore, miR-205 was shown to suppress lung metastasis in an animal model.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, 825 N. Rutledge, PO Box 19626, Springfield, IL 62794, USA.

ABSTRACT
MicroRNAs (miRNAs) are endogenous, small, non-coding RNAs, which are capable of silencing gene expression at the post-transcriptional level. In this study, we report that miR-205 is significantly underexpressed in breast tumor compared to the matched normal breast tissue. Similarly, breast cancer cell lines, including MCF-7 and MDA-MB-231, express a lower level miR-205 than the non-malignant MCF-10A cells. Of interest, ectopic expression of miR-205 significantly inhibits cell proliferation and anchorage independent growth, as well as cell invasion. Furthermore, miR-205 was shown to suppress lung metastasis in an animal model. Finally, western blot combined with the luciferase reporter assays demonstrate that ErbB3 and vascular endothelial growth factor A (VEGF-A) are direct targets for miR-205, and this miR-205-mediated suppression is likely through the direct interaction with the putative miR-205 binding site in the 3'-untranslated region (3'-UTR) of ErbB3 and VEGF-A. Together, these results suggest that miR-205 is a tumor suppressor in breast cancer.

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Identification of ErbB3 and VEGF-A as direct targets for miR-205A, Alignment of miR-205 with ErbB3 at the 3’-UTR. B, Suppression of the luciferase activity by miR-205 in a dose-dependent manner. C. While miR-205 suppresses the luciferase activity for Luc-ErbB3-UTR, it has no effect on Luc-ErbB3-UTR-d. D, Anti-miR-205 increases the luciferase activity of Luc-ErbB3-UTR in 293T cells which overexpress miR-205. E, Alignment of miR-205 with VEGF-A at the 3’-UTR. F, Effect of miR-205 on the luciferase activity of Luc-VEGF-A-UTR and Luc-VEGF-A-UTR-d. G, Anti-miR-205 increases the luciferase activity of Luc-VEGF-A-UTR in 293T cells which overexpress miR-205. Values in B, C, D, F and G are means of three experiments ± SE. **, p < 0.01. SC, scrambled oligo; Anti, anti-miR-205.
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Figure 5: Identification of ErbB3 and VEGF-A as direct targets for miR-205A, Alignment of miR-205 with ErbB3 at the 3’-UTR. B, Suppression of the luciferase activity by miR-205 in a dose-dependent manner. C. While miR-205 suppresses the luciferase activity for Luc-ErbB3-UTR, it has no effect on Luc-ErbB3-UTR-d. D, Anti-miR-205 increases the luciferase activity of Luc-ErbB3-UTR in 293T cells which overexpress miR-205. E, Alignment of miR-205 with VEGF-A at the 3’-UTR. F, Effect of miR-205 on the luciferase activity of Luc-VEGF-A-UTR and Luc-VEGF-A-UTR-d. G, Anti-miR-205 increases the luciferase activity of Luc-VEGF-A-UTR in 293T cells which overexpress miR-205. Values in B, C, D, F and G are means of three experiments ± SE. **, p < 0.01. SC, scrambled oligo; Anti, anti-miR-205.

Mentions: To understand the molecular mechanisms by which miR-205 inhibits tumor cell growth and cell invasion, we searched for putative miR-205 targets as predicted by the commonly cited programs such as TargetScan4 (11), miRBase Target5 (http://microrna.sanger.ac.uk/targets/v5/), PicTar (34)and miRanda (http://www.microrna.org) (35). This search identified several genes that are likely associated with tumorigenicity, including K-RAS, ESRRG (estrogen-related receptor gamma), Bcl-2, eIF4E, ErbB3 and VEGF-A. Therefore, we constructed luciferase reporters carrying the 3’-UTR with the putative miR-205 binding sites for each of those genes. Luciferase assays indicated that both ErbB3 and VEGF-A gave rise to a significant reduction of the luciferase activity. The importance of ErbB3 is highlighted by the fact that ErbB3 is frequently overexpressed in breast cancer and ErbB2/ErbB3 heterodimer is the most potent oncogenic complex (36). Moreover, it has been reported that ErbB3 inactivation blocks proliferation of breast cancer cells at ErbB2-independent manner (37). As shown in Fig. 5A, the ErbB3-3’UTR carries a conserved miR-205 binding site. Luciferase assays revealed that the reduction of luciferase activity was in a dose-dependent manner (Fig. 5B). To further confirm that miR-205-mediated reduction of the luciferase activity for Luc-ErbB3-3’UTR vector is due to direct interaction between miR-205 and its putative binding site, we deleted the miR-205 binding site by side-directed mutagenesis, resulting in Luc-ErbB3-3’UTR-d. As expected, miR-205-mediated suppression of the luciferase activity was completely abolished in Luc-ErbB3-3’UTR-d (Fig. 5C), suggesting that the miR-205 binding site is critical for its suppression function. To further test the specificity of miR-205-mediated suppression, we used anti-miR-205 oligo. Since 293T cells express a low level of miR-205, we first established stable clones overexpressing miR-205 and then introduced anti-miR-205 into these stable transfectants. As shown in Fig. 5D, anti-miR-205 enhanced its activity by over a 2-fold compared to scrambled oligo.


Suppression of cell growth and invasion by miR-205 in breast cancer.

Wu H, Zhu S, Mo YY - Cell Res. (2009)

Identification of ErbB3 and VEGF-A as direct targets for miR-205A, Alignment of miR-205 with ErbB3 at the 3’-UTR. B, Suppression of the luciferase activity by miR-205 in a dose-dependent manner. C. While miR-205 suppresses the luciferase activity for Luc-ErbB3-UTR, it has no effect on Luc-ErbB3-UTR-d. D, Anti-miR-205 increases the luciferase activity of Luc-ErbB3-UTR in 293T cells which overexpress miR-205. E, Alignment of miR-205 with VEGF-A at the 3’-UTR. F, Effect of miR-205 on the luciferase activity of Luc-VEGF-A-UTR and Luc-VEGF-A-UTR-d. G, Anti-miR-205 increases the luciferase activity of Luc-VEGF-A-UTR in 293T cells which overexpress miR-205. Values in B, C, D, F and G are means of three experiments ± SE. **, p < 0.01. SC, scrambled oligo; Anti, anti-miR-205.
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Figure 5: Identification of ErbB3 and VEGF-A as direct targets for miR-205A, Alignment of miR-205 with ErbB3 at the 3’-UTR. B, Suppression of the luciferase activity by miR-205 in a dose-dependent manner. C. While miR-205 suppresses the luciferase activity for Luc-ErbB3-UTR, it has no effect on Luc-ErbB3-UTR-d. D, Anti-miR-205 increases the luciferase activity of Luc-ErbB3-UTR in 293T cells which overexpress miR-205. E, Alignment of miR-205 with VEGF-A at the 3’-UTR. F, Effect of miR-205 on the luciferase activity of Luc-VEGF-A-UTR and Luc-VEGF-A-UTR-d. G, Anti-miR-205 increases the luciferase activity of Luc-VEGF-A-UTR in 293T cells which overexpress miR-205. Values in B, C, D, F and G are means of three experiments ± SE. **, p < 0.01. SC, scrambled oligo; Anti, anti-miR-205.
Mentions: To understand the molecular mechanisms by which miR-205 inhibits tumor cell growth and cell invasion, we searched for putative miR-205 targets as predicted by the commonly cited programs such as TargetScan4 (11), miRBase Target5 (http://microrna.sanger.ac.uk/targets/v5/), PicTar (34)and miRanda (http://www.microrna.org) (35). This search identified several genes that are likely associated with tumorigenicity, including K-RAS, ESRRG (estrogen-related receptor gamma), Bcl-2, eIF4E, ErbB3 and VEGF-A. Therefore, we constructed luciferase reporters carrying the 3’-UTR with the putative miR-205 binding sites for each of those genes. Luciferase assays indicated that both ErbB3 and VEGF-A gave rise to a significant reduction of the luciferase activity. The importance of ErbB3 is highlighted by the fact that ErbB3 is frequently overexpressed in breast cancer and ErbB2/ErbB3 heterodimer is the most potent oncogenic complex (36). Moreover, it has been reported that ErbB3 inactivation blocks proliferation of breast cancer cells at ErbB2-independent manner (37). As shown in Fig. 5A, the ErbB3-3’UTR carries a conserved miR-205 binding site. Luciferase assays revealed that the reduction of luciferase activity was in a dose-dependent manner (Fig. 5B). To further confirm that miR-205-mediated reduction of the luciferase activity for Luc-ErbB3-3’UTR vector is due to direct interaction between miR-205 and its putative binding site, we deleted the miR-205 binding site by side-directed mutagenesis, resulting in Luc-ErbB3-3’UTR-d. As expected, miR-205-mediated suppression of the luciferase activity was completely abolished in Luc-ErbB3-3’UTR-d (Fig. 5C), suggesting that the miR-205 binding site is critical for its suppression function. To further test the specificity of miR-205-mediated suppression, we used anti-miR-205 oligo. Since 293T cells express a low level of miR-205, we first established stable clones overexpressing miR-205 and then introduced anti-miR-205 into these stable transfectants. As shown in Fig. 5D, anti-miR-205 enhanced its activity by over a 2-fold compared to scrambled oligo.

Bottom Line: In this study, we report that miR-205 is significantly underexpressed in breast tumor compared to the matched normal breast tissue.Of interest, ectopic expression of miR-205 significantly inhibits cell proliferation and anchorage independent growth, as well as cell invasion.Furthermore, miR-205 was shown to suppress lung metastasis in an animal model.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, 825 N. Rutledge, PO Box 19626, Springfield, IL 62794, USA.

ABSTRACT
MicroRNAs (miRNAs) are endogenous, small, non-coding RNAs, which are capable of silencing gene expression at the post-transcriptional level. In this study, we report that miR-205 is significantly underexpressed in breast tumor compared to the matched normal breast tissue. Similarly, breast cancer cell lines, including MCF-7 and MDA-MB-231, express a lower level miR-205 than the non-malignant MCF-10A cells. Of interest, ectopic expression of miR-205 significantly inhibits cell proliferation and anchorage independent growth, as well as cell invasion. Furthermore, miR-205 was shown to suppress lung metastasis in an animal model. Finally, western blot combined with the luciferase reporter assays demonstrate that ErbB3 and vascular endothelial growth factor A (VEGF-A) are direct targets for miR-205, and this miR-205-mediated suppression is likely through the direct interaction with the putative miR-205 binding site in the 3'-untranslated region (3'-UTR) of ErbB3 and VEGF-A. Together, these results suggest that miR-205 is a tumor suppressor in breast cancer.

Show MeSH
Related in: MedlinePlus