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A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation.

Yu Z, Wang C, Wang M, Li Z, Casimiro MC, Liu M, Wu K, Whittle J, Ju X, Hyslop T, McCue P, Pestell RG - J. Cell Biol. (2008)

Bottom Line: The cell cycle effect of miR-17/20 was abrogated by cyclin D1 siRNA and in cyclin D1-deficient breast cancer cells.In summary, these studies identify a novel cyclin D1/miR-17/20 regulatory feedback loop through which cyclin D1 induces miR-17-5p/miR-20a.In turn, miR-17/20 limits the proliferative function of cyclin D1, thus linking expression of a specific miRNA cluster to the regulation of oncogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.

ABSTRACT
Decreased expression of specific microRNAs (miRNAs) occurs in human tumors, which suggests a function for miRNAs in tumor suppression. Herein, levels of the miR-17-5p/miR-20a miRNA cluster were inversely correlated to cyclin D1 abundance in human breast tumors and cell lines. MiR-17/20 suppressed breast cancer cell proliferation and tumor colony formation by negatively regulating cyclin D1 translation via a conserved 3' untranslated region miRNA-binding site, thereby inhibiting serum-induced S phase entry. The cell cycle effect of miR-17/20 was abrogated by cyclin D1 siRNA and in cyclin D1-deficient breast cancer cells. Mammary epithelial cell-targeted cyclin D1 expression induced miR-17-5p and miR-20a expression in vivo, and cyclin D1 bound the miR-17/20 cluster promoter regulatory region. In summary, these studies identify a novel cyclin D1/miR-17/20 regulatory feedback loop through which cyclin D1 induces miR-17-5p/miR-20a. In turn, miR-17/20 limits the proliferative function of cyclin D1, thus linking expression of a specific miRNA cluster to the regulation of oncogenesis.

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The inverse correlation between cyclin D1 abundance and miR-17/20 expression in human breast cancer tissues and cell lines. (A) Western blots showed the repression of cyclin D1 and AIB1 abundance by miR-17/20. Both anti–miR-17-5p and anti–miR-20a increased the expression of cyclin D1 and AIB1 in cells. AIB1 is a positive control. β-tubulin was a loading control. (B) Cotransfection of anti–miR-17-5p or anti–miR-20a reversed the antiproliferative function of miR-17/20 in MCF-7 cells. (C) Western blots showed the high cyclin D1 level in low miR-17/20–expressing breast cancer cell lines, and low cyclin D1 in high miR-17/20 cell lines. A Northern blot of miRNA17/20 is shown in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200801079/DC1). (D) The abundances of cyclin D1 and miR-17-5p/miR-20a in 16 human breast tumor tissues and 16 matching normal breast tissues were determined by Western and Northern blots. Tissue sample IDs were provided by the sample provider. Black lines indicate that intervening lanes have been spliced out. N, normal tissue; T, tumor tissue. (E) Statistically significant down-regulation of miR-17/20 expression in breast tumors over matching normal tissue. P = 0.004 by Wilcoxon signed rank test. The grayscale intensity of each band in D was obtained by AlphaImager software. The y axis value stands for the addition of miR-17-5p and miR-20a expression in each sample. (F) Significant up-regulation of cyclin D1 expression in breast tumors over matching normal tissue. P = 0.001. (G) Plotting the paired difference of tumor and normal samples expression for each marker (miR-17/20 vs. cyclin D1). The exact McNemar's test indicates a significant association between the up-regulation of expression in cyclin D1 and the down-regulation of miR-17/20 expression. P = 0.002.
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fig3: The inverse correlation between cyclin D1 abundance and miR-17/20 expression in human breast cancer tissues and cell lines. (A) Western blots showed the repression of cyclin D1 and AIB1 abundance by miR-17/20. Both anti–miR-17-5p and anti–miR-20a increased the expression of cyclin D1 and AIB1 in cells. AIB1 is a positive control. β-tubulin was a loading control. (B) Cotransfection of anti–miR-17-5p or anti–miR-20a reversed the antiproliferative function of miR-17/20 in MCF-7 cells. (C) Western blots showed the high cyclin D1 level in low miR-17/20–expressing breast cancer cell lines, and low cyclin D1 in high miR-17/20 cell lines. A Northern blot of miRNA17/20 is shown in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200801079/DC1). (D) The abundances of cyclin D1 and miR-17-5p/miR-20a in 16 human breast tumor tissues and 16 matching normal breast tissues were determined by Western and Northern blots. Tissue sample IDs were provided by the sample provider. Black lines indicate that intervening lanes have been spliced out. N, normal tissue; T, tumor tissue. (E) Statistically significant down-regulation of miR-17/20 expression in breast tumors over matching normal tissue. P = 0.004 by Wilcoxon signed rank test. The grayscale intensity of each band in D was obtained by AlphaImager software. The y axis value stands for the addition of miR-17-5p and miR-20a expression in each sample. (F) Significant up-regulation of cyclin D1 expression in breast tumors over matching normal tissue. P = 0.001. (G) Plotting the paired difference of tumor and normal samples expression for each marker (miR-17/20 vs. cyclin D1). The exact McNemar's test indicates a significant association between the up-regulation of expression in cyclin D1 and the down-regulation of miR-17/20 expression. P = 0.002.

Mentions: MCF-7 cells express abundant cyclin D1. In contrast with MCF-7 cells, transduction of MCF-10A and BT-474 cells, which have low abundance of cyclin D1 (Fig. 3), with the miR-17/20 cluster had no demonstratable effect on DNA synthesis induced by FBS (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200801079/DC1). These results indicate that the inhibition of G1/S transition by miR-17/20 is cyclin D1 dependent.


A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation.

Yu Z, Wang C, Wang M, Li Z, Casimiro MC, Liu M, Wu K, Whittle J, Ju X, Hyslop T, McCue P, Pestell RG - J. Cell Biol. (2008)

The inverse correlation between cyclin D1 abundance and miR-17/20 expression in human breast cancer tissues and cell lines. (A) Western blots showed the repression of cyclin D1 and AIB1 abundance by miR-17/20. Both anti–miR-17-5p and anti–miR-20a increased the expression of cyclin D1 and AIB1 in cells. AIB1 is a positive control. β-tubulin was a loading control. (B) Cotransfection of anti–miR-17-5p or anti–miR-20a reversed the antiproliferative function of miR-17/20 in MCF-7 cells. (C) Western blots showed the high cyclin D1 level in low miR-17/20–expressing breast cancer cell lines, and low cyclin D1 in high miR-17/20 cell lines. A Northern blot of miRNA17/20 is shown in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200801079/DC1). (D) The abundances of cyclin D1 and miR-17-5p/miR-20a in 16 human breast tumor tissues and 16 matching normal breast tissues were determined by Western and Northern blots. Tissue sample IDs were provided by the sample provider. Black lines indicate that intervening lanes have been spliced out. N, normal tissue; T, tumor tissue. (E) Statistically significant down-regulation of miR-17/20 expression in breast tumors over matching normal tissue. P = 0.004 by Wilcoxon signed rank test. The grayscale intensity of each band in D was obtained by AlphaImager software. The y axis value stands for the addition of miR-17-5p and miR-20a expression in each sample. (F) Significant up-regulation of cyclin D1 expression in breast tumors over matching normal tissue. P = 0.001. (G) Plotting the paired difference of tumor and normal samples expression for each marker (miR-17/20 vs. cyclin D1). The exact McNemar's test indicates a significant association between the up-regulation of expression in cyclin D1 and the down-regulation of miR-17/20 expression. P = 0.002.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2500136&req=5

fig3: The inverse correlation between cyclin D1 abundance and miR-17/20 expression in human breast cancer tissues and cell lines. (A) Western blots showed the repression of cyclin D1 and AIB1 abundance by miR-17/20. Both anti–miR-17-5p and anti–miR-20a increased the expression of cyclin D1 and AIB1 in cells. AIB1 is a positive control. β-tubulin was a loading control. (B) Cotransfection of anti–miR-17-5p or anti–miR-20a reversed the antiproliferative function of miR-17/20 in MCF-7 cells. (C) Western blots showed the high cyclin D1 level in low miR-17/20–expressing breast cancer cell lines, and low cyclin D1 in high miR-17/20 cell lines. A Northern blot of miRNA17/20 is shown in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200801079/DC1). (D) The abundances of cyclin D1 and miR-17-5p/miR-20a in 16 human breast tumor tissues and 16 matching normal breast tissues were determined by Western and Northern blots. Tissue sample IDs were provided by the sample provider. Black lines indicate that intervening lanes have been spliced out. N, normal tissue; T, tumor tissue. (E) Statistically significant down-regulation of miR-17/20 expression in breast tumors over matching normal tissue. P = 0.004 by Wilcoxon signed rank test. The grayscale intensity of each band in D was obtained by AlphaImager software. The y axis value stands for the addition of miR-17-5p and miR-20a expression in each sample. (F) Significant up-regulation of cyclin D1 expression in breast tumors over matching normal tissue. P = 0.001. (G) Plotting the paired difference of tumor and normal samples expression for each marker (miR-17/20 vs. cyclin D1). The exact McNemar's test indicates a significant association between the up-regulation of expression in cyclin D1 and the down-regulation of miR-17/20 expression. P = 0.002.
Mentions: MCF-7 cells express abundant cyclin D1. In contrast with MCF-7 cells, transduction of MCF-10A and BT-474 cells, which have low abundance of cyclin D1 (Fig. 3), with the miR-17/20 cluster had no demonstratable effect on DNA synthesis induced by FBS (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200801079/DC1). These results indicate that the inhibition of G1/S transition by miR-17/20 is cyclin D1 dependent.

Bottom Line: The cell cycle effect of miR-17/20 was abrogated by cyclin D1 siRNA and in cyclin D1-deficient breast cancer cells.In summary, these studies identify a novel cyclin D1/miR-17/20 regulatory feedback loop through which cyclin D1 induces miR-17-5p/miR-20a.In turn, miR-17/20 limits the proliferative function of cyclin D1, thus linking expression of a specific miRNA cluster to the regulation of oncogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.

ABSTRACT
Decreased expression of specific microRNAs (miRNAs) occurs in human tumors, which suggests a function for miRNAs in tumor suppression. Herein, levels of the miR-17-5p/miR-20a miRNA cluster were inversely correlated to cyclin D1 abundance in human breast tumors and cell lines. MiR-17/20 suppressed breast cancer cell proliferation and tumor colony formation by negatively regulating cyclin D1 translation via a conserved 3' untranslated region miRNA-binding site, thereby inhibiting serum-induced S phase entry. The cell cycle effect of miR-17/20 was abrogated by cyclin D1 siRNA and in cyclin D1-deficient breast cancer cells. Mammary epithelial cell-targeted cyclin D1 expression induced miR-17-5p and miR-20a expression in vivo, and cyclin D1 bound the miR-17/20 cluster promoter regulatory region. In summary, these studies identify a novel cyclin D1/miR-17/20 regulatory feedback loop through which cyclin D1 induces miR-17-5p/miR-20a. In turn, miR-17/20 limits the proliferative function of cyclin D1, thus linking expression of a specific miRNA cluster to the regulation of oncogenesis.

Show MeSH
Related in: MedlinePlus