MUCIN 1 ONCOPROTEIN EXPRESSION IS SUPPRESSED BY THE miR-125b ONCOMIR - PubMed (original) (raw)

MUCIN 1 ONCOPROTEIN EXPRESSION IS SUPPRESSED BY THE miR-125b ONCOMIR

Hasan Rajabi et al. Genes Cancer. 2010.

Abstract

The MUC1 oncoprotein is overexpressed in most human breast cancers by mechanisms that are incompletely understood. The microRNA, miR-125b, is downregulated in breast cancer cells. The present studies demonstrate that the MUC1 3'UTR contains a site for binding of the miR-125b seed region. The results show that the MUC1 3'UTR suppresses luciferase expression and that this effect is abrogated by mutation or deletion of the miR-125b binding site. Expression of an anti-sense miR-125b in BT-549 breast cancer cells was associated with induction of MUC1 protein, but not MUC1 mRNA, levels. The anti-sense miR-125b also increased BT-549 cell growth by a MUC1-dependent mechanism. In addition, overexpression of exogenous miR-125b downregulated MUC1 protein, and not MUC1 transcripts, in ZR-75-1 breast cancer cells. Silencing of MUC1 in ZR-75-1 cells with a siRNA has been shown to promote DNA damage-induced apoptosis. In concert with these observations, miR-125b-induced decreases in MUC1 levels increased the apoptotic response of ZR-75-1 cells to cisplatin treatment. These findings indicate that miR-125b suppresses translation of the MUC1 oncoprotein and that miR-125b thereby functions as a tumor suppressor in breast cancer cells.

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Conflict of interest statement

The authors declared no potential conflicts of interest with respect to the authorship and/or publication of this article.

Figures

Figure 1.

Figure 1.

Human MUC1 3′UTR contains a potential miR-125b binding site. (A) Identification of a potential miR-125b binding sequence in the MUC1 3′UTR at bp 46 to 67. Six bases of the MUC1 sequence are complementary with the miR-125b seed region. Expression of (B) miR-125b and (C) MUC1 mRNA was determined by RT-PCR for the indicated cell lines. (D) Lysates from the indicated cells were immunoblotted with anti-MUC1-C and as a control anti-β-actin.

Figure 2.

Figure 2.

miR-125b interacts with the MUC1 3′UTR. (A) The wild-type MUC1 3′UTR was cloned into the pMIR-luciferase vector (pMIR-LUC/MUC1-WT3′UTR). The indicated MUC1 sequence was then mutated (MUC1-MUT3′UTR) or deleted (MUC1-DEL3′UTR). (B) BT-549 and (C) MCF-10A cells were transfected with the indicated pMIR-LUC vectors and Renilla-LUC. At 72 h after transfection, the cells were analyzed for luciferase activity. The results are expressed as relative luciferase activity (mean ± SD of 3 determinations) as compared to that expressing pMIR-LUC/MUC1-WT3′UTR (assigned a value of 1).

Figure 3.

Figure 3.

Antisense miR-125b promotes MUC1 expression and BT-549 cell growth. (A) BT-549 cells were infected with a control lentivrus (Vector) and one expressing the antisense miR-125b (pMIRZIP-125b). MUC1 expression was determined by quantitative RT-PCR (qRT-PCR), and the results are expressed as the relative 2−ΔCt (mean ± SD of 3 determinations) as compared to that obtained for the vector control (assigned a value of 1) (left). Lysates from the indicated BT-549 cells were immunoblotted with anti-MUC1-C and anti-β-actin (right). (B) The BT-549/vector (open triangles) and BT-549/ZIP-125b (closed squares) cells were seeded at 0.5 × 104 cells/mL. Cell number was determined by Trypan blue staining at the indicated times. (C) BT-549/vector and BT-549/ZIP-125b cells were seeded in 6-well plates (1,000 cells per well). Colonies were stained with crystal violet at 14 days and photographed. (D) BT-549/ZIP-125b cells were transfected with a control siRNA and a MUC1siRNA for 48 h. Lysates were immunoblotted with the indicated antibodies (left). Growth of the cells transfected with the control siRNA (closed diamonds) and MUC1siRNA (closed squares) was assessed by Trypan blue staining at the indicated times (right).

Figure 4.

Figure 4.

miR-125b downregulates MUC1 expression and sensitizes ZR-75-1 cells to DNA damage-induced apoptosis. (A) ZR-75-1 cells were infected with a control lentivirus (Vector) and one expressing miR-125b. miR-125b levels were assessed by RT-PCR. (B) MUC1 expression in the indicated ZR-75-1 cells was determined by quantitative RT-PCR (qRT-PCR), and the results are expressed as the relative 2−ΔCt (mean ± SD of 3 determinations) as compared to that obtained for the vector control (assigned a value of 1) (left). Lysates from the indicated cells were immunoblotted with anti-MUC1-C and anti-β-actin (right). (C) ZR-75-1/vector and ZR-75-1/miR-125b cells were left untreated (Control) and treated with 25 µM CDDP for 72 h. Cells were stained with propidium iodide and analyzed for cell cycle distribution by flow cytometry. (D) ZR-75-1/vector (shaded bars) and ZR-75-1/miR-125b (solid bars) cells were treated with the indicated concentrations of CDDP for 72 h. The cells were analyzed by flow cytometry for sub-G1 DNA. The results are expressed as the percentage apoptosis (mean ± SD for 3 experiments).

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