The present study was approved by the Ethics Committee of Kunming Medical University (The Third Affiliated Hospital of Kunming Medical University, Kunming, China) and informed consent was obtained from all patients. Breast cancer tissue and normal tissue (>5 cm distant from cancer tissue) were collected from patients who were pathologically diagnosed with breast cancer at the Tumor Hospital of Yunnan Province from December 2015 to March 2016. None of the patients underwent any therapy prior to specimen collection, including chemotherapy and radiotherapy. Collected tissue samples were immediately stored in RNAlater (Tiangen Biotech Co., Ltd., Beijing, China) at −80°C. A total of 19 female patients with breast cancer were recruited into the present study (age range, 39–68 years; mean average age, 53 years). The clinicopathological characteristics of each patient were recorded, including age, tumor size, Tumor-Node-Metastasis (TNM) stage, estrogen receptor (ER) status, progesterone receptor (PR) status and lymph node involvement.

The human triple negative breast cancer cell line, HCC1937, and the mammary gland cell line, MCF-10A, were donated by Professor Ceshi Chen from the Kunming Institute of Zoology (Kunming, China), CAS (Chinese Academy of Sciences). was recovered from cryopreservation in liquid nitrogen (−196°C) and used at an early passage. 293 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). HCC1937 cells were maintained in RPMI-1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences), 10 U/ml penicillin and 0.1 mg/ml streptomycin (HyClone; GE Healthcare Life Sciences). MCF-10A cells were maintained in Dulbecco's Modified Eagle's medium (DMEM)-F12 medium (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS, 10 U/ml penicillin and 0.1 mg/ml streptomycin. 293 cells were maintained in Dulbecco's Modified Eagle's medium (DMEM; HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS, 10 U/ml penicillin and 0.1 mg/ml streptomycin. HCC1937, MCF-10A and 293 cells were then incubated at 37°C in 5% CO₂.

Tissue miRNA was extracted using the miRcute miRNA isolation kit (Tiangen Biotech Co., Ltd.), according to the manufacturer's protocol. cDNA was synthesized using the miRcute Plus miRNA First-strand cDNA Synthesis kit (Tiangen Biotech Co., Ltd.). The thermocycling conditions were as follows: 60 min at 42°C and 3 min at 95°C. miR-199b-5p expression was analyzed using the miRcute Plus miRNA qPCR Detection kit (Tiangen Biotech Co., Ltd.) with the 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The thermocycling conditions were as follows: An initial pre-denaturation step of 15 min at 95°C, followed by 40 cycles of denaturation at 94°C for 20 sec and annealing at 60°C for 34 sec. The primers sequences are as follows miR-199b-5p, forward, 5′-CAGCCCAGTGTTTAGACTATC-3′ and reverse, 5′-CAGTGCAGGGTCCGAGGT-3′, and U6, forward, 5′-CTCGCTTCGGCAGCACATATACT-3′ and reverse, 5′-ACGCTTCACGAATTTGCGTGTC-3′. Relative expression was calculated using the 2^−∆∆Cq method (23). The RT-qPCR assays were performed in duplicate and the data were presented as the mean ± standard error of the mean. Using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) for statistical analysis, the relative quantity (RQ) value was defined as the ratio of miR-199b-5p expression in target samples to that in control samples. An RQ <1 and ≥1 indicated low and high expression of miR-199b-5p in the target samples, respectively.

The miR-199b-5p mimic and negative control (NC) miRNA were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The miR-199b mimics sequence was 5′-CCCAGUGUUUAGACUAUCUGUUC-3′ and the NC sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. The miR-199b-5p mimic and NC (40 nM) were transfected into cells using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) in serum-free conditions for 6 h prior to replacement with complete medium. Western blotting and RT-qPCR were performed 48 h post-transfection.

To assess the mechanism by which miR-199b-5p inhibits cell proliferation and migration, an miRNA target search was performed using TargetScan (http://www.targetscan.org). The results demonstrated that the 3′UTR of DDR1 contained a highly conserved putative miR-199b-5p binding site. Luciferase reporter plasmids, pRL-DDR1-3′UTR wild type (WT) and pRL-DDR1-3′UTR mutant (MUT) plasmids were synthesized and verified by Shanghai Genomeditech Co., Ltd (Shanghai, China). For the luciferase reporter assay, 293 cells were co-transfected with pRL-DDR1-3′UTR WT or pRL-DDR1-3′UTR MUT, and miR-199b-5p mimics or NC using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The pRL-TK plasmid (Promega Corporation, Madison, WI, USA) was used as control. The successful overexpression vector was incubated with LB (Luria-Bertani) liquid medium overnight at 37°C and the plasmid was prepared using a high purity Plasmid Extraction kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions. The pRL-TK 3′-UTR target plasmid (10 ng) was transiently transfected into 293 cells, using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) with a pRL-TK 3′-UTR negative control plasmid (10 ng). Results were obtained according to the protocol of the reportable gene detection kit (Shanghai Genomeditech Co., Ltd., Shanghai, China) and luciferase activities were analyzed using a Dual Luciferase Reporter assay system (Promega Corporation, Madison, WI, USA) 48 h after transfection. Firefly luciferase activity was used for normalization.

Total protein from cells was extracted using 1% radioimmunoprecipitation lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) containing phenylmethanesulfonylfluoride (Beijing Solarbio Science & Technology Co., Ltd.). The miR-199b-5p mimic group was transfected with miR-199b-5p mimics, the negative control group transfected with negative control miR, the control group was untreated. The protein concentration of total cell lysates was measured using a BCA protein assay kit (Tiangen Biotech Co., Ltd.). Proteins (20 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MD, USA). Membranes were blocked at room temperature with 5% non-fat milk for 1 h and incubated at 4°C overnight with the following antibodies: DDR1 (dilution, 1:500; cat. no. ENT1307; Elabscience, Wuhan, China) and GAPDH (dilution, 1:3,000; cat. no. 21612; Signalway Antibody LLC, College Park, MD, USA). Membranes were then washed with TBST three times for 10 min, incubated with a goat anti-rabbit immunoglobulin G secondary antibody (dilution, 1:3,000; cat. no. L3012; Signalway Antibody LLC) for 2 h at room temperature and washed with Tris-buffered saline with Tween 3 times for 10 min. The protein was detected by chemiluminescence using an ECL detection reagent (Beijing Solarbio Science & Technology Co., Ltd.). Finally, the quantification of western blotting was performed using Image J software (version 1.51; National Institutes of Health, Bethesda, MD, USA).

HCC1937 cells were transfected with the miR-199b-5p mimic and negative control for 48 h. A marker pen and a ruler were first used to draw 5 lines on each well of a 6-well culture dish. Subsequently, 2×10⁶ HCC1937 cells cultured for 12 h at 37°C in 5% CO₂, and then a 200-µl pipette tip was used to scratch two or more parallel wounds of ~200 mm in width, perpendicular to the marker lines. Wounds were observed following incubation at 37°C in 5% CO₂ for 12 and 24 h under a phase contrast microscope (magnification, ×100) to assess healing rate.

The miR-199b-5p mimic and negative control were transfected into HCC1937 cells as aforementioned. Transwell chambers without and with Matrigel (Corning Incorporated, Corning, NY, USA) were used to assess cell migration and invasion, respectively. A total of 5×10⁴ cells in 200 µl RPMI-1640 medium without FBS were placed into the cell culture inserts (BD Biosciences Japan, Co., Ltd, Tokyo, Japan). A total of 600 µl RPMI-1640 medium containing 10% FBS was added to the bottom chamber. Cells were then incubated for 24 h at 37°C in 5% CO₂, fixed with paraformaldehyde and stained with 0.1% crystal violet at room temperature for 20 min. Cell migration and invasion were analyzed by counting the cells on the underside of the membrane using a light microscope (magnification, ×100) in 5 random fields of view.

The effect of miR-199b-5p on the proliferation of HCC1937 cells was assessed using a CCK-8 assay. Cells in 200 µl of DMEM were seeded into 96-well plates (2×10³/well) and incubated at 37°C in 5% CO₂ for 48 h. Cell proliferation was evaluated every 12 h for 1 day. A total of 10 µl CCK-8 (Biosharp, Hefei, China) was added to each well and incubated for an additional 4 h at 37°C in 5% CO₂. A CCK8 Enzyme-linked immune monitor (Thermo Fisher Scientific, Inc.) was utilized to assess absorbance values at a wavelength of 450 nm.

Statistical analysis was performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard error of the mean and were analyzed using a Student's t-test (between two groups) or one-way analysis of variance followed by Tukey's test (multiple groups). The expression of miR-199b-5p and clinicopathological parameters were analyzed using a χ² test. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using GraphPad Prism 7.0a Macintosh Version (GraphPad Software Inc., La Jolla, CA, USA).

The TNBC cell line, HCC1937, exhibited the lower expression of miR-199b-5p compared with the MCF-10A cell line (Fig. 1A). To determine the effect of miR-199b-5p on HCC1937 cell migration and invasion, cells were treated with a miR-199b-5p mimic and negative control miR. Observation with fluorescence microscope (Fig. 1B) and the results of RT-qPCR assay (Fig. 1C) demonstrated that the relative expression of miR-199b-5p was significantly elevated in the mimic group compared with the negative control. A wound-healing assay demonstrated that the miR-199b-5p mimic significantly decreased the migration rate of HCC1937 cells (Fig. 2A) compared with the negative control. Furthermore, the Transwell migration assay revealed that miR-199b-5p mimics significantly decreased the number of migrated cells compared with the negative control group. The effect of the miR-199b-5p mimic on HCC1937 cell invasion was assessed using a Matrigel invasion assay, which demonstrated similar results to the migration assay (Fig. 2B).

To assess the role of miR-199b-5p in the proliferation of HCC1937 cells, a gain-of-function approach was utilized. HCC1937 cells were transfected with a miR-199b-5p mimic and negative control miR. Furthermore, cell proliferation was evaluated using a CCK-8 assay at different time points. It was revealed that the upregulation of miR-199b-5p significantly inhibited HCC1937 cell proliferation at days 3–5 in comparison with the control group (Fig. 3A).

A total of 19 samples were obtained from patients with breast cancer who did not receive any treatment prior to surgery. Breast cancer tissues and normal adjacent breast tissue (>5 cm from the lesion) were excised. Compared with normal breast tissues, the expression of miR-199b-5p in breast cancer tissues was significantly decreased (Fig. 3B). Furthermore, there was no significant association between miR-199b-5p expression and any assessed characteristics, including age, tumor size and TNM stage were observed (Table I).

The expression of miR-199b-5p and DDR1 in breast cancer and the association between them were analyzed using public databases (http://starbase.sysu.edu.cn). Compared with normal breast tissue, the expression of miR-199b-5p in breast cancer tissues was significantly reduced (Fig. 4A). These results are congruent with those that were obtained from the present study. Furthermore, compared with normal breast tissue, the expression of DDR1 in breast cancer tissues was significantly upregulated (Fig. 4B). The expression of miR-199b-5p was also demonstrated to be negatively correlated to DDR1 (Fig. 4C).

To assess the mechanism by which miR-199b-5p inhibits cell proliferation and migration, an miRNA target search was performed using TargetScan (http://www.targetscan.org). The results demonstrated that the 3′UTR of DDR1 contained a highly conserved putative miR-199b-5p binding site. Western blotting also revealed that the expression of DDR1 was significantly decreased in the miR-199b-5p mimic group compared with the negative control group and the control group (Fig. 5A). In addition, pRL-DDR1 3′UTR-wild type (WT) and DDR1 pRL-3′UTR-mutant (MUT) plasmids were generated to assess the interaction between miR-199b-5p and its putative target sequences. The results of the luciferase reporter assay demonstrated that the luciferase activity of DDR1 3′UTR-luc-WT was decreased upon transfection with the miR-199b-5p mimic, compared with those transfected with negative control (Fig. 5B). These results indicated that miR-199b-5p directly inhibited DDR1 expression by binding to the 3′UTR of DDR1.

Taken together, the data of the present study indicate that miR-199b-5p serves as a potent oncogenic miRNA in HCC1937 cells by affecting cell proliferation, migration and invasion, possibly by targeting DDR1.