Unregulated miR-96 induces cell proliferation in human breast cancer by downregulating transcriptional factor FOXO3a

Abstract

FOXO transcription factors are key tumor suppressors in mammalian cells. Until now, suppression of FOXOs in cancer cells was thought to be mainly due to activation of multiple onco-kinases by a phosphorylation-ubiquitylation-mediated cascade. Therefore, it was speculated that inhibition of FOXO proteins would naturally occur through a multiple step post-translational process. However, whether cancer cells may downregulate FOXO protein via an alternative regulatory mechanism is unclear. In the current study, we report that expression of miR-96 was markedly upregulated in breast cancer cells and breast cancer tissues compared with normal breast epithelial cells (NBEC) and normal breast tissues. Ectopic expression of miR-96 induced the proliferation and anchorage-independent growth of breast cancer cells, while inhibition of miR-96 reduced this effect. Furthermore, upregulation of miR-96 in breast cancer cells resulted in modulation of their entry into the G1/S transitional phase, which was caused by downregulation of cyclin-dependent kinase (CDK) inhibitors, p27(Kip1) and p21(Cip1), and upregulation of the cell-cycle regulator cyclin D1. Moreover, we demonstrated that miR-96 downregulated FOXO3a expression by directly targeting the FOXO3a 3'-untranslated region. Taken together, our results suggest that miR-96 may play an important role in promoting proliferation of human breast cancer cells and present a novel mechanism of miRNA-mediated direct suppression of FOXO3a expression in cancer cells.

Figure 1. Upregulation of miR-96 promoted proliferation of breast cancer cells.

A, Real-time PCR analysis of miR-96 expression in normal breast epithelial cells (NBECs) and breast cancer cell lines, including BT549, ZR-75-30, Bcap37, MDA-MB231, MDA-MB435, MCF-7, SKBR3, MDA-MB453 and T47D. B, The expression of miR-96 was examined in 15 paired breast tumor tissues (tumor) and their adjacent normal tissues (normal). The average miR-96 expression was normalized by U6 expression. Each bar represents the mean of three independent experiments. C, Effects of miR-96 overexpression on the growth of breast cancer cells MCF-7 and ZR-75-30. MTT assays revealed that miR-96-transfected cells proliferated more rapidly than the vector control cells. D, Representative micrographs (left) and quantification (right) of crystal violet stained cell colonies. E, Quantification of Ki-67 positive cells in MCF-7 and ZR-75-30 cells transfected with miR-96 mimic and negative control (NC). F, Upregulation of miR-96 promoted breast cancer cell tumorigenicity as determined by anchorage-independent growth assay. Representative micrographs (left) and quantification of colonies that contained more than 50 cells (middle) or were larger than 0.1 mm (right) were scored. Each bar represents the mean of three independent experiments. * P<0.05.

Figure 2. miR-96 induced proliferation through increasing the proportion of S phase cells.

A, Flow cytometric analysis of indicated breast cancer cells transfected with miR-96 mimic or NC. B, Representative micrographs (left) and quantification of BrdU incorporating-cells after transfection with miR-96 or NC. C, Western blotting analysis of expression of p21^Cip1, p27^Kip1, cyclin D1, phosphorylated Rb (p-Rb) and total Rb protein in indicated cells. α-tubulin was used as a loading control. D, Real-time PCR analysis of expression of p21^Cip1, p27^Kip1, cyclin D1 in indicated cells. GAPDH was used as a loading control. Error bars represent mean ± SD from three independent experiments. * P<0.05.

Figure 3. Inhibition of miR-96 suppressed the proliferation of breast cancer cells.

A, Real-time PCR analysis of expression of p21^Cip1, p27^Kip1 and cyclin D1 in indicated cells. GAPDH was used as a loading control. Error bars represent mean ± SD from three independent experiments. B, Flow cytometric analysis of indicated breast cancer cells transfected with miR-96 inhibitor or NC. C, MTT assays revealed that inhibition of miR-96 reduced cell growth. D, Representative micrographs (left) and quantification of colonies that contained more than 50 cells (middle) or were larger than 0.1 mm (right) determined by anchorage-independent growth assays. Each bar represents the mean of three independent experiments. * P<0.05.

Figure 4. miR-96 downregulated FOXO3a via directly targeting the FOXO3a 3′UTR.

A, Predicted miR-96 target sequences in 3′-UTR of FOXO3a (FOXO3a) and mutant containing two mutated nucleotides in 3′-UTR of FOXO3a (FOXO3a-mu). B, Western blotting analysis of FOXO3a expression in indicated cells. C, Relative FOXO3a reporter activities in the indicated cell lines. D, Western blotting analysis of GFP expression in indicated cells. E, Luciferase assays on MCF-7 or ZR-75-30 breast cancer cells transfected with the pGL3 control reporter, pGL3-FOXO3a-3′UTR reporter, or pGL3-FOXO3a-3′UTR-mu reporter and increasing amounts of miR-96 mimic oligonucleotides (20, 50 nM), as indicated. Error bars represent mean ± SD from three independent experiments. * P<0.05.

Figure 5. Inhibition of miR-96 activated the FOXO3a pathway.

A, Luciferase activity assay of indicated cells transfected with the pGL3-FOXO3a-3′UTR reporter with increasing amounts (20, 50 nM) of miR-96 mimic- or miR-96 inhibitor-oligonucleotides. B, Western blotting analysis of FOXO3a expression in indicated cells. C, Relative FOXO3a reporter activities in the indicated cell lines. Error bars represent mean ± SD from three independent experiments. * P<0.05.

Figure 6. FOXO3a plays an important role in miR-96-induced proliferation.

A, The expression of p21^Cip1, p27^Kip1 and cyclin D1 in indicated cells using real-time PCR analysis. GAPDH was used as a loading control. B, Relative FOXO3a reporter activity in the indicated cell lines. C, The growth rate of breast cancer cells transfected with miR-96 mimic, or mixed miR-96 and FOXO3a, or mixed miR-96 and FOXO3a-3′-UTR. D, Real-time PCR analysis of expression of miR-96, p27 and cyclin D1. E, Expression of FOXO3a, Ki67 (upper) and correlation (lower) analysis of miR-96, FOXO3a, Ki-67 in two normal human breast tissues and eight freshly prepared human breast cancer tissues. * P<0.05.