The human studies were approved by the ethics committee of Yinzhou People's Hospital of Ningbo City (Ningbo, China). Written informed consent was obtained from all patients. A total of 30 pairs of breast cancer and paracarcinoma tissues (≥5 cm away from the tumor edge) were obtained from patients with breast cancer that had not undergone radiotherapy and chemotherapy (age range, 25-55 years old). The patients were admitted to the Yinzhou People's Hospital of Ningbo City between January 2015 and March 2017. After tissue collection, the specimens were fixed at 4°C overnight in 10% formalin for storage. The normal breast epithelial cell line MCF-10A, and the breast cancer cell lines, MCF-7, T47D and MDA-MB-231, were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Roswell Park Memorial Institute-1640 medium supplemented with 10% fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

RT-qPCR was performed to investigate the correlation between miR-194-5p and SOX17 in breast cancer. Once cells reached 60% confluence, TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was applied for RNA extraction. The same method was used for RNA extraction from tissues. RNA concentration was determined using a NanoDrop 2000 instrument (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). M-MLV RT (cat no. 28025-013; Invitrogen; Thermo Fisher Scientific, Inc.) and GoTaq^® DNA Polymerase (cat no. M3005; Promega Corporation, Madison, WI, USA) were used for cDNA synthesis and PCR, respectively. cDNA synthesis was conducted at 95°C for 12 min, and the PCR thermocycling conditions were as follows: 97°C for 5 min, followed by 35 cycles at 95°C for 30 sec, 65°C for 30 sec and 73°C for 1 min, and a final step at 73°C for 10 min, after which samples were maintained at 4°C until use. The miR-194-5p and SOX17 PCR primer sequences were as follows: miR-194-5p, forward, 5'-ACA CTC CAG CTG GGT GTA ACA GCA ACT CC-3' and reverse, 5'-TGG TGT CGT GGA GTC G-3'; and SOX17, forward, 5'-GTG GAC CGC ACG GAA TTT G-3' and reverse, 5'-GGA GAT TCA CAC CGG AGT CA-3'. The expression levels were normalized to U6 or GAPDH, the primer sequences were as follows: U6, forward, 5'-AGTA AGC CCT TGC TGT CAG TG-3' and reverse, 5'-CCT GGG TCT GAT AAT GCT GGG-3'; and GAPDH, forward, 5'-ACA ACT TTG GTA TCG TGG AAG G-3' and reverse, 5'-GCC ATC ACG CCA CAG TTT C-3'. The 2^−ΔΔCq method was used for relative quantification (17).

MCF-7 cells (1×10⁵) were seeded into 24-well plates, and the miR-194-5p inhibitor (miR20000460-1-5) and miR-194-5p overexpression plasmids (miR10000460-1-5) were obtained from Guangzhou RiboBio Biotechnology Co., Ltd. (Guangzhou, China). The overexpression plasmid was used for luciferase reporter assays. Briefly, 500 ng plasmids were transfected into cells using 1 µl Lipo6000™ Transfection Reagent (Beyotime Institute of Biotechnology, Shanghai, China); the cells were incubated at 37°C in an atmosphere containing 5% CO₂ for 6 h. Subsequently, the transfected cells were incubated for a further 72 h. The empty vector was transfected into cells in the overexpression negative control (NC) groups. The following sequences were transfected for miR-194-5p overexpression: Forward, 5'-CAG GAG TTG TAA ATC CGA GCC G-3' and reverse, 5'-TTC ATA GGT CAG AGC CCT GTG CA-3'.

After 72 h incubation, the MTT assay was performed for cell proliferation analysis. Briefly, MCF-7 cells (5×10⁵ cells/well) were placed into 96-well plates and the cell proliferation rate was determined at various time points (24, 48 and 72 h). Briefly, 20 µl MTT reagent (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to cells and incubated for 4 h at 37°C. Subsequently, formazan crystals were dissolved in dimethyl sulfoxide and the optical density (OD) value was detected at 490 nm using a microplate reader.

The colony formation assay was performed after transfection and 72 h incubation. Cells (1,500 cells/well) were plated into 12-well plates and cultured for 10 days to form colonies. The cells were then fixed with methanol for 15 min at 4°C and stained with Giemsa dye solution for 30 min at room temperature to visualize cell colonies. Images were captured under a light microscope.

Post-transfection, the migration and invasion of MCF-7 cells was evaluated by scratch and Transwell assays. For the scratch assay, MCF-7 cells in log phase were seeded into 96-well plates to obtain a monolayer cell culture. Across the center of the well, the cell monolayer was scratched using a fresh 1-ml pipette tip; the width of the scratch was equal to the outer diameter of the tip. Subsequently, the cells were incubated at 37°C in an atmosphere containing 5% CO₂ for 72 h. Images of the migrated cells were captured after 24 h under an inverted microscope (CKX41; Olympus Corporation, Tokyo, Japan) and were analyzed using ImageJ v1.8.0 (National Institutes of Health, Bethesda, MD, USA). For the Transwell assay, Transwell culture inserts (pore size, 8 mm; Falcon; BD Biosciences, Franklin Lakes, NJ, USA) were placed into the wells of 96-well plates, thus leading to separated upper and lower chambers. The upper side of the membrane was precoated with Matrigel (BD Biosciences) and incubated at 37°C for 1 h for gel formation. FBS was used to hydrate the membrane 2 h prior to use. Subsequently, Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.; 600 µl) containing 10% FBS was added to the lower chamber, whereas 1×10⁵ cells/well were added to the upper chamber. After 72 h at 37°C, the number of invading cells was counted using a counting chamber under an inverted microscope (CKX41; Olympus Corporation).

Western blot analysis was conducted after 72 h incubation. Proteins were isolated from transfected MCF-7 cells using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) and protein concentration was measured using a bicinchoninic acid kit (Beyotime Institute of Biotechnology). Cytosolic protein was extracted using the Cytosol Protein Extraction kit (#P0033; Beyotime Institute of Biotechnology). Nuclear protein was extracted using the Nucleoprotein Extraction kit (#C500009-0050; Sangon Biotech Co., Ltd., Shanghai, China). Subsequently, the protein samples (20 µg/per lane) were separated by 15% SDS-PAGE. Polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA) were used for protein transfer. Subsequently, the PVDF membranes were blocked with 5% skimmed milk for 2 h at room temperature, and were incubated with the following primary antibodies: Anti-SOX17 (#81778, 1:2,000), anti-Wnt (#2915, 1:2,000), anti-β-catenin (#8480, 1:500), anti-phosphorylated (p)-β-catenin (#9567, 1:500) and anti-GAPDH (#5174, 1:2,000) (Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature, followed by incubation with secondary antibodies (sc-2004, 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 45 min at room temperature. An enhanced chemiluminescence kit (#32209; Pierce; Thermo Fisher Scientific, Inc.) was applied for visualization and the images were analyzed using ImageJ v1.8.0 (National Institutes of Health).

Bioinformatics analysis with TargetScan, and luciferase reporter assays were conducted to verify that SOX17 was a direct target gene of miR-194-5p. For luciferase reporter assays, MCF-7 cells were seeded into 96-well plates at a density of 2×10⁵ cells/well and grown to 70% confluence. Following the implementation of site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis kit (Guangzhou RiboBio Biotechnology Co., Ltd.), cells were co-transfected with miR-194-5p mimic or miR-194-5p NC for 48 h at 37°C (50 ng; Guangzhou RiboBio Biotechnology Co., Ltd.), and SOX17-3' untranslated region (UTR)-wild-type (WT) (50 ng) or SOX17-3'UTR-mutant (MUT) (50 ng) plasmids (Guangzhou RiboBio Biotechnology Co., Ltd.) using Lipo6000™ Transfection Reagent (0.2 µl). Furthermore, transfection efficiency was normalized to a Renilla luciferase vector (pRL-CMV; Promega Corporation). The luciferase assay kit (BioLux^® Gaussia; New England Biolabs, Inc., Ipswich, MA, USA) was used to evaluate lucif-erase activity according to the manufacturer's protocol.

The animal studies were approved by the laboratory animal management and welfare ethical review committee of Yinzhou People's Hospital of Ningbo City. A total of 18 female BALB/c-nu/nu nude mice (age, 4-5 weeks; weight, 18-25 g) were obtained from Zhejiang Experimental Animal Center. The mice were housed in a specific pathogen-free laboratory and were maintained under the following conditions: Constant temperature, 22-26°C; humidity, 40-70%; 12-h light/dark cycle; free access to food and water). Briefly, the mice were randomly divided into groups A and B; mice in group A were used for tumor weight and volume analysis, whereas mice in group B were used for immunohistochemical analysis. Briefly, 500 ng miR-194-5p inhibitor and negative control (NC) were transfected into MCF-7 cells with 1 µl Lipo6000™ Transfection Reagent at 37°C with 5% CO₂ for 6 h. Subsequently, 1×10⁷/0.3 ml MCF-7 cells in the log phase were inoculated into the left side of the breast to generate a murine model of breast cancer. After 20 days ad libitum feeding, the tumor-burdened nude mice exhibited tumors ~0.5 cm in diameter. Mice were divided into the control, NC and miR-194-5p inhibitor groups.

Tumor weight was measured using an electric scale and tumor volume was recorded using a vernier caliper 20 days after MCF-7 cell inoculation.

After 20 days ad libitum feeding, the mice were anesthetized and sacrificed. Subsequently, the tumor tissues were obtained and fixed in 10% formalin at 4°C overnight. Sections were incubated with anti-SOX17 primary antibodies (#81778S, 1:1,000; Cell Signaling Technology, Inc.) for 1 h at room temperature, after which they were incubated with secondary antibodies (#7074, 1:2,000; Cell Signaling Technology, Inc.) for 10 min at room temperature. Images of staining were captured under an inverted microscope (CKX41; Olympus Corporation) and were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Each experiment was repeated in triplicate and all data are presented as the means ± standard deviation. Additionally, Student's t-test was used for two group comparisons, whereas one-way analysis of variance followed by the Dunnett's post hoc test was used for multiple group comparisons, and the Pearson's correlation coefficient was employed for correlation analysis. Data were analyzed using SPSS 14.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to investigate the correlation between miR-194-5p and SOX17 in breast cancer, the expression levels of miR-194-5p and SOX17 were determined by RT-qPCR. As shown in Fig. 1A and B, miR-194-5p was significantly upregulated, whereas SOX17 was markedly downregulated in breast cancer tissues compared with in normal tissues. In addition, a negative correlation was detected between miR-194-5p and SOX17 (r²=0.8907; Fig. 1C), thus indicating that the expression of miR-194-5p was negatively correlated with SOX17 in breast cancer. It is of great significance to further elucidate the correlation between miR-194-5p and SOX17, and the regulatory relationship of the Wnt signaling pathway.

The expression levels of miR-194-5p were also detected in numerous breast cancer cell lines, in order to select an appropriate cell line for subsequent experiments. As shown in Fig. 2, miR-194-5p was significantly increased in MCF-7, T47D and MDA-MB-231 cells compared with in MCF-10A cells. Furthermore, miR-194-5p was most upregulated in MCF-7 cells; therefore, MCF-7 was chosen as the target cell line in the present study.

The expression levels of miR-194-5p were measured by RT-qPCR, and the protein expression levels of SOX17 and Wnt in transfected MCF-7 cells were determined by western blotting. As shown in Fig. 3A, the expression levels of miR-194-5p were markedly decreased in the miR-194-5p inhibitor group compared with in the control groups. Furthermore, the expression levels of SOX17 and Wnt were upregulated in the miR-194-5p inhibitor-transfected group compared with in the control groups (Fig. 3B and C). Therefore, knockdown of miR-194-5p in MCF-7 cells may promote the expression of SOX17.

The cytosolic and nuclear protein expression levels of p-β-catenin and β-catenin were also evaluated by western blotting. The results demonstrated that p-β-catenin was significantly upregulated in the cytosol and nucleus in the miR-194-5p inhibitor group compared with in the control groups. However, there was no difference in the expression levels of β-catenin in the cytosol and nucleus between the miR-194-5p inhibitor group and the control groups (Fig. 4). Therefore, knockdown of miR-194-5p in MCF-7 cells might facilitate the expression of p-β-catenin in the cytosol and nucleus, with no effects on β-catenin expression.

The MTT assay was performed to assess proliferation of miR-194-5p inhibitor-transfected MCF-7 cells. The proliferation rate was determined by OD values. As shown in Fig. 5A, proliferation was significantly decreased in the miR-194-5p inhibitor group compared with in the control groups. As presented in Fig. 5B and C, the proliferation rate was further determined by colony formation assay. The results indicated that proliferation was significantly decreased in the miR-194-5p inhibitor group. These results suggested that knockdown of miR-194-5p may suppress the proliferation of MCF-7 cells.

The migration and invasion of transfected MCF-7 cells were measured by scratch and Transwell assays, respectively. As shown in Fig. 6A and B, cell migration and invasion were significantly inhibited in the miR-194-5p inhibitor group compared with in the control groups. Therefore, knockdown of miR-194-5p in MCF-7 cells might inhibit cell migration and invasion.

The results of a TargetScan analysis suggested that SOX17 was a target gene of miR-194-5p, due to a complementary binding region in its 3'UTR. A luciferase reporter assay was performed to verify this prediction. As shown in Fig. 7, luciferase activity was decreased in the SOX17 3'UTR WT group transfected with miR-194-5p mimics, thus suggesting that SOX17 may be a direct target gene of miR-194-5p.

A total of 20 days after injection with miR-194-5p inhibitor-transfected cells, tumor growth was measured according to tumor volume and weight. As shown in Fig. 8, tumor volume and weight were significantly decreased in the miR-194-5p inhibitor group compared within the control groups. These findings indicated that knockdown of miR-194-5p may contribute to tumor growth suppression in breast cancer.

SOX17 expression was measured by IHC after injection with miR-194-5p inhibitor-transfected cells for 20 days. The results indicated that the expression levels of SOX17 were markedly increased in the miR-194-5p inhibitor group compared with in the control groups (Fig. 9). Therefore, knockdown of miR-194-5p may give rise to the upregulation of SOX17 in breast cancer tissues.