miR‐516a‐3p inhibits breast cancer cell growth and EMT by blocking the Pygo2/Wnt signalling pathway

Abstract miR‐516a‐3p has been reported to play a suppressive role in several types of human tumours. However, the expression level, biological function and fundamental mechanisms of miR‐516a‐3p in breast cancer remain unclear. In the present study, we found that miR‐516a‐3p expression was down‐regulated and Pygopus2 (Pygo2) expression was up‐regulated in human breast cancer tissues and cells. Through analysing the clinicopathological characteristics, we demonstrated that low miR‐516a‐3p expression or positive Pygo2 expression was a predictor of poor prognosis for patients with breast cancer. The results of a dual luciferase reporter assay and Western blot analysis indicated that Pygo2 was a target gene of miR‐516a‐3p. Moreover, overexpression of miR‐516a‐3p inhibited cell growth, migration and invasion as well as epithelial‐mesenchymal transition (EMT) of breast cancer cells, whereas reduced miR‐516a‐3p expression promoted breast cancer cell growth, migration, invasion and EMT. Furthermore, we showed that miR‐516a‐3p suppressed cell proliferation, metastasis and EMT of breast cancer cells by inhibiting Pygo2 expression. We confirmed that miR‐516a‐3p exerted an anti‐tumour effect by inhibiting the activation of the Wnt/β‐catenin pathway. Finally, xenograft tumour models were used to show that miR‐516a‐3p inhibited breast cancer cell growth and EMT via suppressing the Pygo2/Wnt signalling pathway. Taken together, these results show that miR‐516a‐3p inhibits breast cancer cell growth, metastasis and EMT by blocking the Pygo2/ Wnt/β‐catenin pathway.

regulation. 3 Emerging evidence shows that miRNAs participate in many biological processes, such as cell proliferation 4 and apoptosis. 5 A large number of studies have demonstrated that miRNAs can either promote the progression of cancer or act as tumour suppressors. 6,7 miRNAs also play crucial roles in tumour cell migration, invasion and epithelial-mesenchymal transition (EMT). [8][9][10][11][12][13] The cancer-related miRNA miR-516a-3p has been shown to be down-regulated in gastric cancer 14 and ovarian cancer, 15 suggesting it has a role in suppressing human cancers. Yoshifumi Takei et al 14

demonstrated
that the expression of miR-516a-3p in 44As3 cells (highly metastatic gastric cancer cells) was lower than that in HSC-44PE cells (parental cells isolated from patients), and sulfatase 1 was a direct target of the miR-516a-3p in gastric cancer. In vivo, miR-516a-3p markedly decreased metastases to the peritoneum. NMA White et al 15 confirmed that miR-516a-3p can target kallikrein 10 (KLK10) and subsequently affect ovarian cancer cell proliferation. In this study, we investigated the role of miR-516a-3p in breast cancer for the first time.
Epithelial-mesenchymal transition has been found to be closely related to tumour invasion and metastasis. 16,17 The molecular mechanisms of EMT are complex, and various molecules have been found to regulate EMT, including miRNAs. [18][19][20][21] Pygo2 has been shown to promote the proliferation of breast cancer cells. 22 Zhang S et al 23 reported that Pygo2 promoted cell invasion and metastasis through decreasing E-cadherin expression in hepatic carcinoma. Additionally, Pygo2 is a novel functional protein, downstream of the Wnt signalling pathway. 24 Pygo2 primarily binds to free β-catenin to promote the progression of cancer by activating β-catenin target genes, including cyclin D1 and c-Myc. 22,25 Interestingly, the activation of the Wnt/β-catenin signalling pathway has been reported to accelerate EMT in bladder cancer cells. 26 In this study, we report for the first time the role of miR-516a-3p breast cancer. We demonstrated that miR-516a-3p inhibited breast cancer cell growth, metastasis and EMT by blocking the Pygo2/ Wnt/β-catenin signalling pathway both in vitro and in vivo.  and MCF-7 cells were seeded into 6-well plates and cultured for 24 hours at 37°C with 5% CO 2 . Lipofectamine 3000 reagent was diluted with Opti-MEM. miR-516a-3p mimic, miR-516a-3p inhibitor, pcDNA3.1-Pygo2 plasmid or Pygo2 siRNA were dissolved with Opti-MEM, added into diluted Lipofectamine 3000 and incubated for 5 minutes at room temperature. Subsequently, the cells were added with the mixtures and serum-free medium.

| RNA extraction and qRT-PCR
RNA was extracted from tissue samples or cells using TRIzol Reagent (TaKaRa) following the manufacturer's instructions. A Mir-X miRNA First-Strand Synthesis Kit (TaKaRa) and PrimeScript RT Master Mix (TaKaRa) were used for reverse transcription. qRT-PCR was performed using SYBR Premix Ex Taq (Roche) to detect the relative expression of miR-516a-3p and Pygo2. The specific primers used were synthesized by Sangon ( Table 1). The 2 (−ΔΔCT) method was used to calculate the level of miR-516a-3p and Pygo2 expression. U6 and GAPDH were regarded as controls for miR-516a-3p and Pygo2, respectively.

| Immunohistochemical staining and scoring
Tissues were paraffin-embedded and sliced into 4-μm thick sections for

| Western blotting
Proteins were extracted from breast cancer tissues or cells using RIPA lysis buffer supplemented with protease inhibitor. Next, total proteins were separated using gel electrophoresis and transferred to polyvinylidene antimouse IgG (1:10 000, Affinity) and goat anti-rabbit IgG (1:10 000, Affinity), for 1 hour. ECL detection reagent (Santa Cruz) was added on the membranes to detect signals. GAPDH used as a loading control. The greyscale values of protein bands were analysed using ImageJ.

| Proliferation assay
Cells were passed in 96-well plates at a density of 2000 cells/well after transfection. Cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay. The principle of the CCK-8 assay is the same as that of the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. 29 However, because the formazan pro-  with Annexin V and PI. Samples were analysed using a flow cytometer.

| Wound healing assay
Wound healing assay was used to detect the migration of MDA-

MB-231 and MCF-7 cells. Transfected cells were cultured in 6-well
plates until the conflux reached 80%. The cell layer in each well was wounded by scratching the cells with aseptic 10 μL plastic pipette tips. Images of the scratches were captured at 0 and 48 hours.

| TUNEL
Apoptotic cells in the breast cancer tumour sections from mice were detected via a TUNEL assay. Sections were dewaxed in xylene for 5-10 minutes and then were dewaxed in fresh xylene for 5-10 minutes. Sections were placed in absolute ethanol for 5 minutes, 95% ethanol for 2 minutes, 85% ethanol for 2 minutes, 75% ethanol for 2 minutes and distilled water for 2 minutes. Next, the tissues were incubated with 2% DNase-free proteinase K at 37°C for 20 minutes, washed them with PBS three times and then were blocked with 5% serum at 37°C for 20 minutes. The sections were treated following the manufacturer's instructions for the In Situ Cell Death Detection Kit (Roche Applied Science). Images were acquired under a fluorescence microscope (Germany Lecia photomicrography system) and Image-Pro Plus (Media Cybernetics) was used to evaluate positive staining in the sections.

| Statistical analysis
SPSS (version 21.0; SPSS, Inc) was used to analyse the data. The miRNA expression and Pygo2 expression in paired tumour and adjacent normal tissues were determined using the paired two-sample Student's t test. The data among the groups were detected by the Student's t test or a one-way analysis of variance (ANOVA) and shown as the means ± standard deviation. Correlations between clinicopathological parameters and miR-516a-3p or Pygo2 expression were analysed with chi-squared test. Survival analysis was determined using Kaplan-Meier plots and log-rank tests. Differences with P < 0.05 were regarded as significance.
F I G U R E 2 Pygo2 is a target gene of miR-516a-3p. A, miR-516a-3p and its putative binding sequence in the 3′-UTR of Pygo2 mRNA. Mutant miR-516a-3p binding sites were generated in the complementary site for the seed region of miR-516a-3p (WT, wild type; Mut, mutant type). B, miR-516a-3p effects on luciferase activity in cells that carried the wild

| miR-516a-3p expression is down-regulated and inversely correlated with Pygo2 expression in human breast cancer tissue and cell lines
To assess the expression level of miR-516a-3p and Pygo2 in breast cancer, we detected their expression in 60 paired breast cancer tissue and matched normal breast tissue samples. qRT-PCR results showed that miR-516a-3p expression was significantly down-regulated in most of the breast cancer tissue samples compared with that in the matched controls ( Figure 1A). IHC staining results showed that Pygo2 protein expression was up-regulated in 68% (41/60) of the breast cancer tissue samples (Table 2, Figure 1B). In the cell lines, we found miR-516a-3p expression was lower in breast cancer cells MCF-7 and MDA-MB-231 than that in the normal breast cell line HBL-100 ( Figure 1C), whereas Pygo2 protein and mRNA expression were higher in breast cancer cells MCF-7 and MDA-MB-231 than that in the normal breast cells HBL-100 ( Figure 1D-E). These data show that the miR-516a-3p expression is down-regulated and Pygo2 expression is up-regulated in breast cancer.

| Low miR-516a-3p expression or positive Pygo2 expression is a predictor of poor prognosis for patients with breast cancer
As shown in Table 2, the low level of miR-516a-3p expres- Pygo2 was also related to lymph node metastasis (P = 0.025), increased tumour size (P = 0.041) and worse TNM stage (P = 0.037).
In both luminal A and basal-like of breast cancer, the expression of miR-516a-3p was lower and positive rate of Pygo2 expression was higher than that in adjacent normal breast tissues. However, there was no significant correlation between the expression of miR-516a-3p/Pygo2 and molecular subtypes (P > 0.05, Table 2).
Furthermore, compared with patients with high miR-516a-3p levels, patients with low miR-516a-3p level had shorter overall survival (OS; P < 0.05, Figure 1F). Additionally, the OS for patients with positive Pygo2 expression was markedly worse than for those patients with negative Pygo2 expression (P < 0.05, Figure 1G). These results indicate that lower miR-516a-3p expression or positive Pygo2 expression is related to poor clinical features in breast cancer patients. These results show that both miR-516a-3p and Pygo2 have the potential to be prognostic biomarkers for breast cancer.
In order to further explore the relationship between miR-516a-3p and Pygo2, MCF-7 and MDA-MB-231 cells were transfected with miR-516a-3p mimic or miR-516a-3p inhibitor to up-regulate or down-regulate miR-516a-3p expression, respectively. qRT-PCR analysis showed that miR-516a-3p mimic and miR-516a-3p inhibitor suc-  Figure 2F). IHC staining results showed that the level of Pygo2 protein in breast cancer tissues exhibiting high miR-516a-3p expression was lower than that in breast cancer tissues with low miR-516a-3p expression. (Figure 2G) These data indicate that Pygo2 is a target gene of miR-516a-3 in breast cancer.

| miR-516a-3p inhibits migration, invasion and EMT of breast cancer cells
To confirm the suppressive metastasis role of miR-516a-3p, we evaluated cell migration and invasion using wound healing and transwell assays. The results showed that migration and invasion were significantly inhibited after cells were transfected with miR-516a-3p mimic compared with that of the control group ( Figure 4A, 4). On the other hand, migration and invasion were significantly increased after breast cancer cells were treated with miR-516a-3p inhibitor ( Figure 4B, 4). These data show that miR-516a-3p inhibits breast cancer cell migration and invasion.
Epithelial-mesenchymal transition is a crucial process for tumour invasion and metastasis. To investigate the effect of miR-516a-3p on EMT, MCF-7 and MDA-MB-231 cells were transfected with miR-516a-3p mimic, miR-516a-3p inhibitor or negative control.
Western blot results demonstrated that miR-516a-3p overexpression induced increased E-cadherin protein expression and decreased vimentin protein expression ( Figure 4E). On the other hand, we found that miR-516a-3p reduction decreased E-cadherin protein expression and increased vimentin protein expression ( Figure 4F).
These results indicate that miR-516a-3p inhibits EMT in breast cancer, and further confirm that it is a suppressor of metastasis in breast cancer cells.

| miR-516a-3p inhibits cell proliferation, migration, invasion and EMT by blocking Pygo2 expression in breast cancer cells
We further investigated the mechanism of miR-516a-3p inhibit-  Figure 5H).
These results demonstrate that miR-516a-3p inhibits cell proliferation and EMT through blocking Pygo2 expression in breast cancer.

| miR-516a-3p inhibits activity of the Wnt/βcatenin pathway of breast cancer cells
Pygo2 is a novel functional protein downstream of the Wnt signalling pathway which is also related to EMT. To further study whether miR-516a-3p has an effect on the Wnt pathway, we detected key molecules in the Wnt signalling pathway. miR-516a-3p overexpression markedly reduced β-catenin, c-Myc and cyclin D1 expression ( Figure 6A), while miR-516a-3p depletion enhanced β-catenin, c-Myc and cyclin D1 expression ( Figure 6B). These data indicate that miR-516a-3p inhibits activity of the Wnt/β-catenin pathway.

| miR-516a-3p inhibits breast cancer cells growth and EMT by blocking the Pygo2/Wnt signalling pathway in vivo
To explore the effect of miR-516a-3p on breast cancer cell growth in vivo, MDA-MB-231 cells were inoculated into the right-side fat pads of female BALB/c nude mice. After 6 days, the tumours of mice were separately injected into PBS, angomir control and miR-516a-3p angomir every other day. After 3 weeks, the tumours injected with the miR-516a-3p angomir showed potent tumour growth inhibition ( Figure 7A-B) with the weights of the tumours in miR-516a-3p angomir group being lower than those in angomir control group and blank group ( Figure 7C).
Western blot analysis showed that E-cadherin expression was significantly increased by miR-516a-3p angomir compared with angomir control and PBS. Vimentin, Pygo2, β-catenin, c-Myc, and cyclin D1 expression was obviously decreased by miR-516a-3p angomir compared with angomir control and PBS ( Figure 7D). Additionally, the TUNEL assay results showed that the apoptosis rate of breast cancer tumour tissue increased after miR-516a-3p angomir treatment compared with angomir control and PBS ( Figure 7E). IHC staining results showed that E-cadherin protein was up-regulated and Pygo2 protein and vimentin protein were down-regulated in breast cancer tumour tissues treated with miR-516a-3p angomir ( Figure 7F). These results suggest that miR-516a-3p inhibits breast tumour growth and EMT by blocking the Pygo2/Wnt signalling pathway in vivo.

| D ISCUSS I ON
Breast cancer is one of the most common malignancies, and metastasis is the major cause of death in breast cancer patients. Multiple reports have demonstrated the role of miRNAs in the breast cancer metastasis. 19,[30][31][32] In this study, the results demonstrate that miR-516a-3p inhibits breast cancer cells growth, metastasis and EMT by suppressing the Pygo2/Wnt/β-catenin signalling pathway ( Figure 7G).
In this study, we found that miR-516a-3p expression was de- Our results demonstrated that miR-516a-3p inhibited breast cancer cell migration and invasion. miR-516a-3p overexpression increased E-cadherin expression and decreased vimentin expression, and reduced levels of miR-516a-3p decreased E-cadherin expression and increased vimentin expression relative to that in the control groups.
The in vivo Western blot and IHC staining results were consistent with the in vitro. These results indicate that miR-516a-3p is a suppressor of metastasis and EMT in breast cancer.
Overexpression of Pygo2 enhanced hepatic carcinoma cell invasion and metastasis through reducing E-cadherin expression.
To explore the role of Pygo2 in the process of EMT regulated by miR-516a-3p, we increased Pygo2 expression through transfecting cells with pcDNA3.1-Pygo2 plasmid. We found that increasing Pygo2 expression could reverse the effect of miR-516a-3p on the proliferation, migration, invasion and EMT of breast cancer cells.
These results indicate that miR-516a-3p may inhibit cell proliferation, EMT and metastasis through blocking Pygo2.
It has been proved EMT is enhanced by the activation of the Wnt/β-catenin signalling pathway 26,44 and abnormal expression of β-catenin is closely related to the progression, metastasis and prognosis of breast cancer. 45 Pygo2 combines with free β-catenin to cause abnormal activation of downstream target genes, including c-Myc and cyclin D1. 22,25 Downregulation of β-catenin by siRNA inhibits cell proliferation and induces apoptosis. 46 Studies have confirmed that c-Myc and cyclin D1 were highly expressed in breast cancer. 47,48 Our data verified that miR-516a-3p suppressed the activity of the Wnt pathway by reducing β-catenin, c-Myc and cyclin D1 protein expression both in vitro and in vivo. We also found that miR-516a-3p induced cell apoptosis in vitro. These results demonstrate that miR-516a-3p inhibits breast cancer cell growth and metastasis through blocking the Wnt/β-catenin pathway.
In this study, we explored the expression level, biological function and fundamental mechanisms of miR-516a-3p in breast cancer for the first time. We found that Pygo2 was a target of miR-516a-3p, and miR-516a-3p suppressed breast cancer cell growth and metastasis by inhibiting the Pygo2/Wnt/β-catenin pathway, which were never reported. Either miR-516a-3p expression or Pygo2 expression was not related to molecular subtypes of breast cancer, but they were therapeutic targets and potential prognostic biomarkers of breast cancer.
In summary, we confirmed that the expression of miR-516a-3p was decreased and that of Pygo2 was increased in human breast cancer. The reduction of miR-516a-3p expression and the overexpression of Pygo2 were related to poor clinical features and a worse prognosis in breast cancer patients. Moreover, miR-516a-3p suppressed cell growth and EMT through blocking Pygo2 and inhibiting the Wnt pathway both in vitro and in vivo. These results provide insight into the molecular mechanism of miR-516a-3p in breast cancer cell growth and metastasis and demonstrate that miR-516a-3p may be a novel therapeutic target to treat breast cancer progression and metastasis.

ACK N OWLED G EM ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.