In total 35 pairs of BC tissues and matched adjacent non-tumor tissues were obtained from patients (age, 28–65 years; 35 females) who were surgically treated at the Breast Center, The Fourth Hospital of Hebei Medical University (Shijiazhuang, China) between August 2020 and November 2021. These patients were all pathologically diagnosed with breast cancer. None of the patients received any treatment prior to surgery and written informed consent was obtained from all patients. The clinicopathological information of the patients is presented in Table I. Tissue samples were frozen in liquid nitrogen and stored in a refrigerator at −80°C. The present study was approved by the Ethics Committee of The Fourth Hospital of Hebei Medical University (approval no. 2021KY056). The present study was conducted in accordance with the principles described in The Declaration of Helsinki.

The human breast epithelial MCF10A cell line and BC SK-BR3, MCF7 and BT549 cell lines were purchased from Procell Life Science & Technology, Co., Ltd. Cells were cultured in RPMI-1640 media (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated with 5% CO₂ at 37°C. miR-217-5p mimic, miR-negative control (NC) mimic, miR-217-5p inhibitor and miR-NC-inhibitor were synthesized by Shanghai GenePharma Co., Ltd., and transfected into SK-BR3 or BT549 cells at a final concentration of 100 nM using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 6 h. Subsequent experimentation was performed 48 h after transfection. The following sequences were used: miR-217-5p mimic, 5′-UACUGCAUCAGGAACUGAUUGGA-3′; miR-NC mimic, 5′-UUGUCCGAACGUGUCACGU-3′; miR-217-5p inhibitor, 5′-UCCAAUCAGUUCCUGAUGCAGUA-3′; and miR-NC inhibitor, 5′-CAGCUGGUUGAAGGGGACCAAA-3′.

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from BC tissues and BC cell lines, including miRNA. RNA quality was assessed using the A260/A280 ratio. This value was determined using a microplate reader (Multiskan Sky; Thermo Fisher Scientific, Inc.). RNA quantity was also assessed using a microplate reader. Total RNA was reverse transcribed into complementary (c)DNA using a Transcriptor First Strand cDNA Synthesis Kit [Roche Diagnostics (Shanghai) Co., Ltd.] using the following temperature protocol according to the manufacturer's instructions: 37°C for 15 min and 85°C for 5 sec. The volume of each RT reaction system was 20 µl, including 1 µg of total RNA. DNase was used to remove genomic DNA interference. Subsequently, qPCR was performed to examine gene expression using a SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 5 min; followed by 40 cycles of denaturation at 95°C for 5 sec, 60°C for 30 sec and 72°C for 30 sec, with a final extension step at 72°C for 2 min. The 2^−ΔΔCq method was used to calculate the relative expression levels using GAPDH or U6 as the internal reference gene (29). The primer sequences used for RT-qPCR are presented in Table II.

Cell proliferation was analyzed using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) assay. Briefly, transfected SK-BR3 or BT549 cells were seeded into 96-well plates at a density of 2×10³ cells/well. Following incubation at 37°C for 24, 48 and 72 h, 10 µl CCK-8 solution was added into each well and incubated at 37°C for 4 h. The absorbance of each well at a wavelength of 450 nm was assessed using a microplate reader (Multiskan Sky; Thermo Fisher Scientific, Inc.).

Colony formation assays were performed to detect cell proliferation. Transfected SK-BR3 and BT549 cells were seeded at 1×10³ cells/well in six-well plates and incubated with 5% CO₂ at 37°C. After 15 days, colonies were visible to the naked eye. These colonies were fixed with 4% paraformaldehyde for 15 min at ambient temperature and stained with 1% crystal violet (Beyotime Institute of Biotechnology) for 15 min at ambient temperature. The number of colonies (containing >50 cells) was counted microscopically.

After 48 h of transfection, 2×10⁵ SK-BR3 or BT549 cells were transferred into a 35-mm² petri dish, cultured at 37°C and grown to 90% confluence. A wound was then created using a 200-µl pipette tip to scratch the monolayer before any detached cells were washed away with PBS. The remaining cells were cultured in serum-free culture media, and wound closure was then observed. Images of the wounds were taken at 0 and 24 h after the scratch was made using a light microscope (magnification, ×100; Olympus Corporation). Migratory ability was calculated as the mobility ratio using the following formula: Mobility ratio=(scratch width at 24 h-scratch width at 0 h)/scratch width at 0 h.

Total protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology) from transfected SK-BR3 and BT549 cell lines. Protein concentration was determined by BCA assay (Thermo Fisher Scientific, Inc.). Next, the protein lysates (30 µg) were separated using SDS-PAGE on a 10% gel and separated proteins were then transferred onto PVDF membranes (MilliporeSigma). Subsequently, the membranes were incubated with 5% skimmed milk at room temperature for 2 h, before they were incubated with the following primary antibodies against: MTDH (1:2,000; cat. no. ab227981; Abcam), GAPDH (1:10,000; cat. no. 10494-1-Ig; ProteinTech Group, Inc.), p65 (1:2,000; cat. no. 10745-1-Ig; ProteinTech Group, Inc.), p-p65 (1:2,000; cat. no. 3033; Cell Signaling Technology), IκBα (1:2,000; cat. no. 10268-1-Ig; ProteinTech Group, Inc.), E-cadherin (1:2,000; cat. no. 20874-1-Ig; ProteinTech Group, Inc.), Vimentin (1:20,000; cat. no. 10366-1-Ig; ProteinTech Group, Inc.) and snail family transcriptional repressor 2 (slug; 1:2,000; cat. no. 12129-1-AP; ProteinTech Group, Inc.), overnight at 4°C. After washing with TBST (0.05% Tween 20), the membranes were incubated with the HRP-conjugated goat polyclonal anti-rabbit IgG secondary antibody (1:10,000; cat. no. SA00001-2; ProteinTech Group, Inc.) for 1 h. Protein expression was determined by enhanced chemiluminescence (MilliporeSigma).

After transfection with miR-217-5p mimic, miR-NC mimic, miR-217-5p inhibitor and miR-NC inhibitor for 48 h at 37°C, a total of 5×10⁵ SK-BR3 and BT549 cells were resuspended in 200 µl serum-free medium and subsequently seeded into the upper chamber of a Transwell insert (Corning, Inc.) precoated with 1 µg/µl Matrigel (BD Biosciences) overnight at 37°C. The lower chamber was filled with 500 µl basal medium with 10% FBS to stimulate cell invasion. Following 48 h of cell culture at 37°C, the cells that did not cross the membrane were wiped with a cotton swab, whereas the cells adhering to the lower surface of the membrane were stained with 0.1% crystal violet solution at room temperature for 10 min. The number of invading cells was counted in five randomly selected fields using a light microscope (magnification, ×200; Olympus Corporation).

A total of 5×10⁵ transfected SK-BR3 and BT549 cells were resuspended in 200 µl serum-free medium and were subsequently seeded into the upper chamber of a Transwell insert without Matrigel. All other procedures were performed as described for the invasion assay.

The TargetScan (http://www.targetscan.org/vert_70/) database was used to predict the target genes of miR-217-5p.

The 3′-untranslated regions (UTR) of wild type or mutant MTDH were inserted into the pmirGLO luciferase vector (Shanghai GeneChem Co., Ltd.). These vectors and miR-217-5p mimic or miR-NC mimic were co-transfected into BT549 cells using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h at 37°C. The lysate was cleared and the luciferase reporter activity was assayed at 48 h post-transfection using the Dual-Luciferase Reporter Assay System (Promega Corporation). The firefly luciferase activity was normalized using Renilla luciferase activity.

Data were analyzed using SPSS 23.0 (IBM Corp.) and GraphPad Prism 8 (GraphPad Software, Inc.). Data are presented as the mean ± SD. All comparisons between two groups of cells were performed using unpaired Student's t-tests. Paired Student's t-tests were performed for comparisons between paired tumor and adjacent non-tumor tissues. Two-way ANOVA followed by Bonferroni's post hoc test were used to compare data from the dual-luciferase reporter assay. The gene expression correlation between miR-217-5p and MTDH was assessed using the Pearson's correlation coefficient test. All experiments were performed at least three times. P<0.05 was considered to indicate a statistically significant difference.

miR-217-5p expression levels were demonstrated to be markedly reduced in MCF7, BT549 and SK-BR3 cell lines compared with MCF10A cells (Fig. 1A). Furthermore, the expression levels of miR-217-5p were significantly lower in BC tissues compared with those in the adjacent non-tumor tissues (Fig. 1B). This indicated that miR-217-5p expression levels were lower in both BC cell lines and tissues.

To investigate the specific function of miR-217-5p in BC, miR-217-5p mimic was transfected into SK-BR3 cells, whereas miR-217-5p inhibitor was transfected into BT549 cells (two cell lines with highest transfection efficiency). Compared with the negative control (NC), the results demonstrated that transfection with the miR-217-5p mimic significantly increased miR-217-5p expression levels, whereas miR-217-5p inhibitor transfection significantly decreased miR-217-5p expression levels (Fig. 2A and C). The CCK-8 assay demonstrated that compared with the NC, transfection with the miR-217-5p mimic in SK-BR3 cells significantly inhibited cell proliferation at 48 and 72 h, whereas transfection with the miR-217-5p inhibitor into BT549 cells significantly promoted cell proliferation at 48 and 72 h (Fig. 2B and D). Furthermore, colony formation assays demonstrated that miR-217-5p overexpression resulted in the significant inhibition of BC cell proliferation, whereas miR-217-5p expression knockdown significantly promoted colony formation compared with the NC group (Fig. 2E and F). Wound healing assays determined that transfection with the miR-217-5p inhibitor into BT549 cells significantly promoted cell migration, whereas miR-217-5p overexpression in SK-BR3 cells significantly suppressed cell migration, compared with the NC group (Fig. 2G and H). Transwell assays demonstrated that miR-217-5p knockdown significantly promoted cell migration and invasion in BT549 cells, but miR-217-5p overexpression significantly inhibited SK-BR3 cell migration and invasion compared with the NC group (Fig. 2I and J). The aforementioned results indicated that miR-217-5p exerted a tumor suppressive role in BC cells.

E-cadherin, Vimentin and slug mRNA and protein expression levels were analyzed using RT-qPCR and western blotting to explore the effects of miR-217-5p on SK-BR3 and BT549 cells further. Overexpression of miR-217-5p markedly enhanced E-cadherin mRNA and protein expression levels, whereas Vimentin and slug expression levels were markedly inhibited compared with the NC group (Fig. 3A and C). However, compared with the NC, miR-217-5p knockdown markedly decreased E-cadherin mRNA and protein expression levels but markedly promoted Vimentin and slug mRNA and protein expression levels (Fig. 3B and D). These results suggested that miR-217-5p overexpression may suppress the EMT in BC.

The mRNA and protein expression levels of NF-κB markers were subsequently assessed to determine the effects of miR-217-5p on SK-BR3 and BT549 cells. RT-qPCR and western blotting demonstrated that miR-217-5p overexpression markedly inhibited p65 phosphorylation but markedly promoted IκBα expression levels compared with the NC (Fig. 4A and C). However, miR-217-5p knockdown markedly suppressed IκBα mRNA and protein expression levels but markedly promoted p65 phosphorylation compared with the NC (Fig. 4B and D). These results indicated that the downregulation of miR-217-5p may activate the NF-κB signaling pathway.

TargetScan (http://www.targetscan.org/vert_71/) was used to predict miR-217-5p target genes. MTDH was determined to be a candidate target for miR-217-5p. Therefore, dual-luciferase reporter assays were performed to assess the potential binding between MTDH mRNA and miR-217-5p (Fig. 5A). MTDH mRNA expression levels were demonstrated to be markedly increased in MCF7, BT549 and SK-BR3 cell lines compared with MCF10A cells (Fig. 5B). Moreover, significantly higher MTDH mRNA expression levels were demonstrated in BC tissues compared with adjacent non-tumor tissues (Fig. 5C). Transfection with miR-217-5p mimic significantly reduced the luciferase activity of the wild-type MTDH 3′UTR compared with the miR-NC mimic. However, there was no change in luciferase activity in the mutant-MTDH 3′UTR group (Fig. 5D). Furthermore, MTDH mRNA expression levels were demonstrated to be significantly inversely correlated with those of miR-217-5p in BC tissues (Fig. 5E). The results also demonstrated that compared with the NC, miR-217-5p overexpression markedly inhibited MTDH mRNA and protein expression levels (Fig. 5F), whereas miR-217-5p knockdown markedly promoted MTDH mRNA and protein expression levels (Fig. 5G). These data therefore suggested that miR-217-5p may directly target MTDH and that the expression of miR-217-5p is inversely correlated with MTDH in BC cells.