The human HER2 positive breast cancer cell lines HCC-1954, 21MT1 and JimT1 and human normal breast cell line MCF-10A were bought from the American Type Culture Collection (Manassas, VA, USA). All of the 4 cell lines were cultured in RPMI-1640 medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), which contained 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and incubated at 37°C with 5% CO₂. A total of 35 pairs of the clinical HER2 positive breast cancer and adjacent tissues were collected from 35 patients (age, 21 to 58 years old) who received resection surgery in The Third Affiliated Hospital of Kunming Medical University (Kunming, China) from April 2015 to August 2017. All of the human tissues used in the present study were obtained with written informed consent. The present study was approved by the Ethics Committee of The Third Affiliated Hospital of Kunming Medical University.

Synthetic pre-miR-193a-3p (Shanghai GenePharma Co., Ltd., Shanghai, China) was used to transfect cells to overexpress miR-193a-3p as previously described (24). GRB7 overexpression plasmid was constructed as previously described (25). HCC-1954, 21MT1 and JimT1 cells were plated in 6-well plates (2.5×10⁵ cells/well) and were transfected using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the protocol.

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from all the 3 cell lines with or without treatment with 5-Aza-dc (5 µM) at room temperature for 4 days and human tissues. Universal cDNA Synthesis kit (Exiqon; Qiagen, Inc., Valencia, CA, USA) was used to synthesize first-strand complementary DNA. miRCURY LNA™ Universal RT microRNA PCR (Exiqon; Qiagen, Inc.) was used to conduct qPCR determining miRNA. cDNA synthesis was conducted at 95°C for 12 min, and the qPCR was conducted with the following thermocycling conditions: 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; samples were then kept at 4°C until use. U6 was used as an endogenous control. Primers of hsa-miR-193a-3p (product no. 204591) were obtained from Exiqon. The forward primer of miR-193a-3p was 5′-CTGAGGGCTGGGTCTTTGC-3′ and the reverse primer was 5′-GCCGAGAACTGGGACTTTGT-3′. The forward primer and reverse primer of U6 were 5′-CTCGCTTCGGCAGCACA-3′ and 5′-ACGCTTCACGAATTTGCGT-3′, respectively.

Western blotting was conducted as previously described (26). Antibodies against GRB7 (1:2,000; cat. no. sc-13954; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ERK (1:1,500; cat. no. 4795; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated ERK (1:2,000; cat. no. 4795; Cell Signaling Technology, Inc.), FOXM1 (1:2,000; cat. no. 5436; Cell Signaling Technology, Inc.) and β-actin (1:10,000; cat. no. AC-74; Sigma-Aldrich; Merck KGaA) were used in the present study. The secondary antibody used was horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:1,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Semi-quantitative analysis was performed using ImageJ software v1.8.0 (National Institutes of Health, Bethesda, MD, USA).

Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to measure cell proliferation ability. HCC-1954, 21MT1 and JimT1 cells (3×10³ cells/well) were plated in 96-well culture plates for 72 h. The CCK-8 reagent was added to each well and incubated at 37°C for 1 h. Cell viability was assessed by testing the absorbance at 450 nm using Multiskan MS (Thermo Labsystems, Helsinki, Finland).

HCC-1954, 21MT1 and JimT1 cells (500 cells/well) were plated in 6-well plates and then cultured in complete media for 10 days. After removing media and being washed by ice-cold PBS 2 times, the colonies were fixed with methanol for 15 min at 4°C and stained with crystal violet for 30 min at room temperature. Images were captured using a digital camera (Canon, Inc., Tokyo, Japan).

The wound-healing assay was carried out to measure the migration ability of HER2 positive breast cancer cells. All 3 cell lines (5×10⁵ cells/well) were fused to form a single layer in 6-well plates and a 200-µl sterile pipette tip was used to scratch a single wound on the cell layer. Following 2 rinses with PBS, cells were incubated for another 24 h. The scratch wounds were visualized under an inverted microscope (CKX41; Olympus Corporation, Tokyo, Japan) and the scratch widths were quantified with ImageJ software v1.8.0 (National Institutes of Health).

Transwell invasion assay was performed using Transwell cell invasion assay kits (Corning, Inc., Corning, NY, USA). A total of 3×10⁴ cells were digested and put in the serum-free medium in the upper chamber, with a 2 mg/ml Matrigel-coated membrane containing 8-m pores. The lower Transwell chamber contained Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation for 72 h (37°C, 5% CO₂), the cells were removed from the upper part of the filters by wiping with a cotton swab. Then, cells on the lower surface of the membrane were fixed with 4% formaldehyde at room temperature for 10 min and stained with 0.5% crystal violet for 15 min at room temperature. Finally, the number of invading cells was imaged and counted at ×200 magnification using an inverted microscope (Nikon Corporation, Tokyo, Japan).

The sequence of the GRB7 3′-UTR which is the potential target of miR-193a-3p was ligated into the pmirGLO plasmid (Promega Corporation, Madison, WI, USA). HCC-1954 cells were cotransfected with the pmirGLO-3′-UTR plasmid of the above plasmids or a blank vector using Lipofectamine™ 2000(Invitrogen; Thermo Fisher Scientific, Inc.). The Dual-Luciferase Assay kit (Promega Corporation) was used to conduct luciferase activities, and the transfection efficiency was normalized by co-transfecting with Renilla-luciferase.

A pyrosequencing assay was conducted to detect the percentage of methylation in miR-193a-3p in HER2 positive breast cancer tissues. PSQ Assay Design Software (version 1.0.6; Biotage, Uppsala, Sweden) was used to design the primers used in pyrosequencing analysis.

RIP experiments were performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation kit (EMD Millipore, Billerica, MA, USA) according to the manufacturer's protocol. The co-precipitated RNAs were detected by reverse transcription PCR, as aforementioned. Total RNA (input controls) and normal mouse immunoglobulin G (1:2,000; cat. no. SLM66-0100; Equitech-Bio, Inc., Kerrville, TX, USA) controls were assayed simultaneously to demonstrate that the detected signals were from the RNA that was specifically bound to GRB7 (n=3 for each experiment).

All experiments were repeated at least 3 times independently. Data are presented as the mean ± standard deviation. Two-tailed Student's t-test and one-way analysis of variance followed by Dunnett's C were used to calculate statistically significant differences. All statistical analyses were performed using SPSS software (version 20.0; IBM, Corps., Chicago, IL, USA). P<0.05 was considered to indicate a statically significant difference.

In order to investigate the potential role of miR-193a-3p in HER2 positive breast cancer, the expression of miR-193a-3p in 35 pairs of tumor/adjacent HER2 positive breast cancer tissues were determined and compared. As depicted in Fig. 1A, the expression of miR-193a-3p in tumor tissues was significantly decreased compared with the normal tissues (P<0.001). The expression of miR-193a-3p in HER2 positive breast cancer tissues of different stages and grades was also detected. As presented in Fig. 1B and C, the level of miR-193a-3p decreases significantly with the increase of tumor stage and grade (P<0.001), which means the level of miR-193a-3p in HER2 positive breast cancer is also associated with the malignant degree. Furthermore, the expression of miR-193a-3p in HER2 positive breast cancer cells (3 different cell lines) and normal breast cell was tested by qPCR. The result demonstrated that compared with normal breast cells, miR-193a-3p was significantly downregulated in HER2 positive breast cancer cells (P<0.01; Fig. 1D).

Then the molecular mechanisms underlying the decrease of miR-193a-3p were investigated in HER2 positive breast cancer. According to the usual regulatory mechanism of miRNA in cancer, the alterations of miR-193a-3p expression depending on DNA methylation in HER2 positive breast cancer cells were investigated. After treating with a demethylating agent, 5-Aza-dc (5 µM) for 4 days, qPCR was conducted to detect the expression of miR-193a-3p, which was demonstrated to significantly increase in the 2 tested HER2 positive breast cancer cell lines (P<0.01; Fig. 2A). Subsequently, pyrosequencing analysis demonstrated a significant increase of miR-193a-3p DNA methylation in higher-stage and higher-grade tumors (P<0.01 and P<0.05; Fig. 2B and C, respectively). These results demonstrated that the loss of miR-193a-3p in HER2 positive breast cancer may be caused by DNA hypermethylation.

In order to further investigate the role miR-193a-3p serves during the development of HER2 positive breast cancer, the changes of cell viability, colony formation ability, migration ability and invasion ability of HER2 positive breast cancer cells overexpressing miR-193a-3p were tested. miR-193a-3p mimics were transfected into 3 HER2 positive breast cancer cell lines and its expression was significantly upregulated (P<0.01; Fig. 3A). Through the CCK-8 assay, it was demonstrated that cell proliferation was significantly weakened following 4-6 days of the treatment with miR-193a-3p mimics (P<0.01; Fig. 3B). Colony formation assay demonstrated that miR-193a-3p mimics could significantly reduce the number of cancer cell colonies formed (P<0.01; Fig. 3C). The wound-healing and Transwell assays were carried out to investigate the effect of miR-193a-3p mimics on cell migration and invasion abilities. As Fig. 3D displays, cell migration and invasion abilities of HER2 positive breast cancer cells were significantly suppressed by miR-193a-3p mimics (P<0.01). Moreover, overexpression of miR-193a-3p could inhibit proliferation, migration and invasion of another 2 HER2 positive breast cancer cell lines 21MT1 and JimT1 (Fig. S1). In brief, these findings indicated that the overexpression of miR-193a-3p could inhibit proliferation, migration and invasion of HER2 positive breast cancer cells and further inhibit the development of HER2 positive breast cancer.

The upregulation of miR-193a-3p significantly downregulated the expression of GRB7 at the protein level in all 3 tested HER2 positive breast cancer cells (P<0.01; Fig. 4A and B). Then, a luciferase reporter assay was carried out to investigate if miR-193a-3p could directly target GRB7. As presented in Fig. 4C and D, miR-193a-3p could specifically downregulate wild-type GRB7 but could not affect the expression of miR-193a-3p with a mutant 3′-UTR in HER2 positive breast cancer cells. GRB7 was significantly upregulated in HER2 positive breast cancer tissues (Fig. 4E). The interaction between GRB7 and miR-193a-3p was further confirmed via RIP assays (Fig. 4F). These results suggested that miR-193a-3p could reduce GRB7 through direct targeting its 3′-UTR.

In order to prove if miR-193a-3p suppresses the development of HER2 positive breast cancer through targeting GRB7, the cell viability, colony formation ability, migration ability and invasive ability of HER2 positive breast cancer cells overexpressing miR-193a-3p were tested following overexpressing GRB7. After overexpressing miR-193a-3p, GRB7 in HCC-1954 cells was significantly downregulated (P<0.05), while overexpression of GRB7 could significantly abolish the reduction of GRB7 caused by overexpression of miR-193a-3p (P<0.01; Fig. 5A and B). In HCC-1954 cells overexpressing miR-193a-3p, the overexpression of GRB7 significantly promoted the cell viability, colony formation ability, migration ability and invasive ability (P<0.05; Fig. 5C-F). In conclusion, GRB7 overexpression could abolish the effects on HER2 positive breast cancer cells caused by the overexpression of miR-193a-3p and further promote the development of HER2 positive breast cancer.

According to the close association between the activation of the ERK/FOXM1 signaling pathway and HER2 positive breast cancer, western blotting was conducted to investigate if miR-193a-3p also represses HER2 positive breast cancer through ERK/FOXM1 signaling pathway. Following the over-expression of miR-193a-3p in HCC-1954 cells, the expression of phosphorylated ERK 1/2 and FOXM1 were significantly reduced (P<0.01; Fig. 6A and B). While the overexpression of GRB7 in HCC-1954 cells overexpressing miR-193a-3p could recover the activity of ERK/FOXM1 signaling pathway, which could accelerate HER2 positive breast cancer tumorigenesis (Fig. 6). These findings demonstrated miR-193a-3p may inhibit HER2 positive breast cancer through downregulating GRB7 and inactivating the ERK/FOXM1 signaling pathway.