Introduction

Breast cancer is one of the most commonly detected cancers in women affecting about 1.2 million women worldwide each year [1]. In the past years, despite achieving significant progress in optimization of treatment with better surgery, cytotoxic agents, and endocrine therapy, the prognosis of breast cancer patients have not been altered much [2–5]. Thus, this is urgent to identify the novel molecular makers which can serve as possible therapeutic targets for treatment of breast cancer.

Recently, increasing evidences indicated that microRNAs (miRNAs), non-coding small RNAs, act as oncogenes or tumor suppressors in the development of cancer [6–8]. MiRNAs play important roles in biological progression of many types of human tumors by regulating gene expression through binding to 3′-UTR of the target mRNA [9–11]. Aberrant expression of miRNAs has been implicated in a variety of biological processes related to cancer, including proliferation, apoptosis, migration, and invasion, which act as either tumor oncogenes or suppressors [12–15]. Multiple reports indicated that miR-503 has been showed to be one of the important determinants in cancers [16–20]. However, the relationship between miR-503 and breast cancer remains incompletely understood. In the current study, we found that miR-503 was downregulated in breast cancer tissue and cell lines. Ectopic overexpression of miR-503 in breast cancer cell lines led to the inhibition of cell growth rate and cell cycle progression. Furthermore, we demonstrated that the tumor promoter gene CCND1 is a direct target of miR-503. Taken together, our results indicated that overexpression of miR-503 could inhibit cell proliferation and cell cycle progression in breast cancer by directly suppressing CCND1.

Materials and methods

Clinical specimens

Eight paired human breast cancer tissues and the adjacent non-cancerous tissues (ANT) were obtained from breast cancer patients and histopathologically diagnosed at Breast Disease Center, Department of Surgery, the First Affiliated Hospital, Sun Yat-sen University (Guangzhou, People’s Republic of China). The study was approved by the ethics committee of the First Affiliated Hospital, Sun Yat-sen University (Guangzhou, People’s Republic of China). Written informed consent was obtained from all patients. Tissue samples were collected at surgery, immediately frozen in liquid nitrogen, and stored until total RNAs or proteins were extracted.

Cell culture

Human breast cancer cell lines MCF-7, T47D, MDA-MB-231, BT549, SKBR3, ZR-75-30, and non-malignant breast epithelial cell MCF-3A were purchased from National Rodent Laboratory Animal Resource (Shanghai, People’s Republic of China) and were grown and cultured in RPMI-1640 (Gibco, USA) supplemented with 10 % fetal bovine serum (FBS, Sigma, St Louis, MI), 100 units/ml of penicillin-streptomycin (Invitrogen, Carlsbad, CA). All cells were cultured in a humidified incubator at 37 °C in an atmosphere of 95 % air and 5 % CO2.

Plasmids and transfection

MiR-503 mimic, miR-503 inhibitor, and negative control were purchased from GeneCopoeia (Guangzhou, China). MCF-7 and MDA-MB-231 cells were seeded into six-well plates 24 h before transfection to ensure 60–70 % confluence at the time of transfection. The Lipofectamine 2000 Reagent (Invitrogen, USA) was used for transfections following the manufacturer’s instructions.

RNA extraction and real-time quantitative PCR

MircoRNAs were extracted from cultured cells or patient samples with RNAiso kit for small RNA (Takara, China) according to the manufacturer’s instructions and reversely transcribed into cDNA with the One Step PrimeScript miRNA cDNA Synthesis kit (Takara, China). The resulting cDNAs were quantified with SYBR Green (Takara, China) using an ABI 7500 Fast Real-time PCR System (Applied Biosystems, USA). The relative miR-503 expression levels after normalization to U6 small nuclear RNA were calculated using 2−[(Ct of miR-503) − (Ct of U6)].

Western blotting

All the cells were gathered and lysed in cell lysis buffer 48 h after the transfection. Equal quantities of protein were separated by 10 % SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5 % non-fat milk for 2 h and incubated overnight with primary antibodies including rabbit polyclonal anti-CCND1 (1:1000; Cell Signaling Technology), anti-p21 (1:1000; Cell Signaling Technology), anti-phosphorylated retinoblastoma (anti-p-Rb, 1:1000; Cell Signaling Technology) and anti-pRb (1:1000; Cell Signaling Technology), and anti-β-actin (1:1000; Cell Signaling Technology). After being washed in TBS-T three times, PVDF membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody at a 1:5000 dilution for 2 h. Immunocomplexes were visualized using the chemiluminescence (GE, USA) following the manufacturer’s protocol.

MTT assay

MCF-7 and MDA-MB-231 cells were seeded in 96-well plates with 3 × 103 cells/well 24 h prior to the transfection of miR-503 or miR-503-in or relative control mimics (NC) and assayed 1, 2, 3, 4, 5, and 6 days after transfection. One hundred microliters 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (0.5 mg/ml, Sigma, St Louis, MI) were added into each well and the incubation continued at 37 °C for 4 h, and then, supernatant was removed and 150 μl dimethyl sulfoxide (DMSO) was added to each well to dissolve the precipitate. The absorbance at 490 nm was measured in a Thermo Scientific Multiskan (Thermo Fisher Scientific, USA).

Colony formation assay

MCF-7 and MDA-MB-231 cells were plated six-well plates at a density of 500 cells per well and incubated for 14 days. Colonies were fixed with 10 % formaldehyde for 10 min and stained with 0.1 % crystal violet for 10 min. The number of colonies, defined as >50 cells/colony were counted.

Cell cycle assay

Forty-eight hours after transfection, MCF-7 and MDA-MB-231 cells were harvested in ice-cold phosphate-buffered saline (PBS) and then fixed with 70 % ice-cold ethanol. The fixed cells were incubated with 50 μg/ml propidium iodide (Sigma–Aldrich) and 50 μg/ml RNase A for 30 min at 37 °C in the dark and analyzed by FACScan (Becton Dickinson, USA).

Luciferase assays

For ectopic expression of CCND1, CCND3 ORFs with 3′-UTR were amplified by PCR and then cloned into pGL3 Vector (Promega) downstream of the Renilla luciferase cDNA. The primers selected were as follows: CCND1-3′UTR-wt-up: 5′-GCTGCGAAGTGGAAAC CATC-3′; CCND1-3′UTR-wt-dn: 5′-CCTCCTTCTGCACACATTTGAA-3′. Cells of 60–70 % confluence in 24-well plates were co-transfected with luciferase reporter vectors and miR-503 expressing vectors, and a 1 ng pRLSV40 Renilla luciferase construct was used for normalization. The relative dual-luciferase activity was assayed 48 h after transfection using the dual-luciferase assay kit according to the manufacturer’s protocol (Promega, Wisconsin, WI, USA).

Statistical analysis

All statistical analyses were analyzed with SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) statistical evaluation for data analysis that was determined by Student’s t test and one-way ANOVA. Differences with P < 0.05 were considered statistically significant.

Result

MiR-503 expression was downregulated in breast cancer tissues and cell lines

We compared the expression of miR-503 in breast cancer cells and patients samples, and we found that all six tested breast cancer cell lines showed significantly downregulated expression of miR-503 compared to the non-malignant breast epithelial cell MCF-3A, and the miR-503 expression was consistently downregulated in the breast cancer tissues compared with adjacent non-cancerous tissues (ANT) (Fig. 1). Taken together, our results suggested that miR-503 was underexpressed in breast cancer and might function as a tumor suppressor.

Fig. 1
figure 1

Expression of miR-503 in human breast cancer cell lines and clinical tissues. a Real-time PCR analysis of miR-503 expression in non-malignant breast epithelial cell MCF-3A and breast cancer cell lines, including MCF-7, T47D, MDA-MB-231, BT549, SKBR3, and ZR-75-30. b Relative miR-503 mRNA expression levels in eight paired human breast cancer tissues (T) and the adjacent non-cancerous tissues (ANT) from the same patient were detected by PCR analysis. Experiments were repeated at least three times (a and b). Each bar represents the mean of three independent experiments. *P < 0.05

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MiR-503 inhibited cell proliferation of breast cancer

To further evaluate the effect of miR-503 on breast cancer growth, we transfected the MCF-7 and MDA-MB-231 cells with miR-503 mimics, miR-503-in, or the respective controls. Relative miR-503 expression was detected by using qRT-PCR and transfection of miR-503 restored its expression, while miR-503-in decreased its expression (Figs. 2a and 3a). In MTT and colon information assays, restoration of miR-503 in MCF-7 and MDA-MB-231 cells resulted in significant suppressing cell proliferation (Fig. 2b, c). In contrast, miR-503-in showed the opposite effect (Fig. 3b, c).

Fig. 2
figure 2

Overexpression of miR-503 inhibited breast cancer cell proliferation. a Validation of miR-503 expression levels after transfection by PCR analysis. b MTT assays revealed that upregulation of miR-503 inhibited growth of MCF-7 and MDA-MB-231 cells. c Quantification of crystal violet-stained cell colonies. d Flow cytometric analysis of the indicated MCF-7 and MDA-MB-231 cells transfected with miR-503 or miR-NC. Each bar represents the mean of three independent experiments. *P < 0.05

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Fig. 3
figure 3

Inhibition of miR-503 promoted breast cancer cell proliferation. a Validation of miR-503 expression levels after transfection by PCR analysis. b MTT assays revealed that inhibition of miR-503 promoted growth of MCF-7 and MDA-MB-231 cells. c Quantification of crystal violet-stained cell colonies. d Flow cytometric analysis of the indicated MCF-7 and MDA-MB-231 cells transfected with miR-503 or miR-NC. Each bar represents the mean of three independent experiments. *P < 0.05

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To further probe the regulatory mechanism of miR-503 inhibiting cell proliferation of breast cancer, we conducted a cell cycle assay. A higher proportion of MCF-7 and MDA-MB-231 cells transfected with miR-503 was in the G0/G1 phase and decreased in S phase compared with those transfected with relative controls, while miR-503-in showed the opposite effect. These results demonstrated that miR-503 arrests the cell cycle in the G0/G1 phase, thus inhibiting cell proliferation and then preventing further malignancy progression.

MiR-503 directly targeted CCND1 by binding to its 3′-UTR

Using TargetScan algorithms, we identified CCND1 as a candidate miR-503 target gene. To determine whether miR-503 could decrease CCND1 expression by targeting the predicted binding site, we inserted the CCND1 3′-UTR into pGL3 plasmid and a mutant of the putative binding site was also prepared (Fig. 4a). As showed in Fig. 4b, co-transfection of the miR-503 expression vector along with the CCND1 3′-UTR luciferase construct caused a significant reduction of luciferase activity as compared with the NC in MCF-7 and MDA-MB-231 cells. Meanwhile, miR-503 had no effect on the luciferase activity of CCND1 3′-UTR-mut type. Taken together, our results demonstrated that CCND1 was a direct target of miR-503 in breast cancer cells.

Fig. 4
figure 4

MiR-503 suppresses CCND1 expression by directly targeting the CCND1 3′-UTR. a Predicted miR-503 target sequence in the 3′-UTR of CCND1 (CCND1-3′-UTR) and positions of three mutated nucleotides (green) in the 3′-UTR of CCND1 (CCND1-3′-UTR-mut). b Luciferase reporter assay of MCF-7 and MDA-MB-231 cells transfected with the pGL3-CCND1-3′-UTR reporter or pGL3-CCND1-3′-UTR-mut reporter and miR-503. *P < 0.05. c Western blotting analysis of protein expression of CCND1, p21, p-pRb, and pRb in MCF-7 and MDA-MB-231 cells. β-Actin was used to serve as the loading control. d Levels of CCND1, p21, and p-pRb proteins were quantified using ImageJ software. β-actin was used as an internal control. The data are presented three independent experiments. *P < 0.05.

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Given that our results indicated miR-503 could influence breast cancer cell proliferation, we examined its functions on the expression level of CCND1 downstream genes which regulate cell proliferation and cell cycle, including p21 and pRb. As showed in Fig. 4c, d, the expression level of the p21 protein was markedly upregulated, while p-pRb was decreased in miR-503 overexpressing cells compared with the negative control cells. In contrast, breast cancer cells transfected with miR-503-in showed the opposite effect. Altogether, our results demonstrated that CCND1 was a bona fide target of miR-503.

Discussion

In the current study, we demonstrated that miR-503 was decreased in breast cancer tissues and six cell lines. Furthermore, we found that ectopic overexpression of miR-503 inhibited the proliferation and cell cycle progression of breast cancer cells by arresting cells in the G0/G1 phase in vitro. Moreover, we demonstrated that miR-503 suppressed CCND1 expression via directly targeting its mRNA 3′-UTR. Taken together, our results suggest that overexpression of miR-503 might play an important role in inhibiting carcinogenesis and progression of breast cancer.

Increasing evidences indicated that microRNAs (miRNAs) are occurring, short, non-coding RNAs that control gene expression by repressing mRNA translation or by inducing mRNA degradation [21–23]. MiRNAs can contribute to tumor growth and progression by modulating the expression of many genes [24–26]. Previous studies have revealed that miR-503 expression is downregulated in several cancers, including osteosarcoma, lung cancer, hepatocarcinoma, and gastric cancer [16, 17, 19, 27]. However, it was uncertain whether dysregulation of miR-503 was associated with the progression of breast cancer. Here, our studies showed that expression of miR-503 was markedly downregulated in breast cancer cells and surgical breast cancer specimens. Gain- and loss-of-function studies indicated overexpression of miR-503 could suppress cell proliferation and colony formation and then induce cell cycle arrest breast cancer cells in the G0/G1 phase, suggesting miR-503 as a candidate tumor suppressor in the pathogenesis of breast cancer.

Using TargetScan algorithms, we identified CCND1 as a candidate miR-503 target gene. CCND1 is a crucial regulator of cell cycle that relates to the development of various cancers, which binds with cyclin-dependent kinases (CDK), which subsequently phosphorylates tumor suppressor protein Rb and then allows the cell cycle to progress through G1 into S phase, resulting in promoting cell proliferation [28–30]. In the present study, we found that CCND1 is a direct target of miR-503, which could downregulate its expression. Furthermore, we demonstrated that miR-503 inhibited breast cancer cell proliferation by binding to the 3′-untranslated region (UTR) of CCND1 mRNA. Further experiment showed that expression of p21 was upregulated and p-pRb were downregulated in MCF-7 and MDA-MB-231 cells transfected with miR-503. Altogether, our results indicated that miR-503 functionally modulates cellular proliferation and cell cycle regulators, p21 and pRb, thus relevant to cell proliferation.

Considered as a whole, the current study revealed that miR-503 operated as a potent tumor suppressor that downregulated the expression of CCND1, and then suppressing the cell proliferation of breast cancer, and this implies miR-503 to be a potential mediator for novel miRNA replacement therapy of breast cancer.