The BC microarray data were extracted from the GSE42072 and GSE41922 datasets in the GEO database (www.ncbi.nlm.nih.gov/geo), which is a public functional genomics information base for microarray gene expression datasets. The GSE42072 dataset contained miRNA expression data from 28 BC samples and 20 matched non-cancer tissue samples from the same BC cases based on the GPL15018 platform (32). The GSE41922 dataset contained miRNA expression data from 32 pre-operative serum samples from patients with BC and 22 healthy volunteer serum samples based on the GPL16224 platform (32). The R language ‘limma’ package (R version 4.1.2; http://www.r-project.org/) was used for analyzing miRNAs with differential expression based on the P<0.001 and |logFC|>2 thresholds. In addition, the data were used to generate a heatmap using OmicShare online platform (https://www.omicshare.com/).

In the pre-experiment, the two groups (BC tissue and normal tissue) were designed to use for estimating sample size. CA153, as a diagnostic marker for BC in clinical work (33), has been used as a standard to estimate the required sample size. After obtaining the mean and standard deviation of CA153 expression levels in BC tissue and normal tissue, the σ²=[(n₁−1)S₁² + (n₂−1)S₂² + … + (n_k−1)S_k²]/(n₁ + n₂ + … + n_k) and n=[(Z_α/2 + Z_β)2σ²(1 + 1/k)]/ε² were used to calculate the minimum sample size. It was revealed that 40 samples would be sufficient for RNA and protein extraction; therefore, 40 BC samples and matched adjacent healthy tissue samples were obtained from patients diagnosed with BC undergoing surgery at the Sichuan Cancer Hospital & Institute (Chengdu, China) between January 2018 and December 2019. After surgical resection, the fresh specimens were immediately stored in liquid nitrogen until they could undergo reverse transcription-quantitative PCR (RT-qPCR). The detailed patient characteristics are described in Table I. The Ethical Committee of the Sichuan Cancer Hospital & Institute approved all studies (approval no. 2020-8748) and patients provided written informed consent.

MCF10A normal human breast epithelial cells, and the human BC cell lines T47D, MCF-7, ZR-75-1, MDA-MB-468, SK-BR-3 and BT-549, were obtained from the American Type Culture Collection. The BC cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% streptomycin-penicillin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere containing 5% CO₂. MCF10A cells were cultured in DMEM-Nutrient Mixture F-12 supplemented with 10% FBS at 37°C in an incubator containing 5% CO₂.

BT-549 and MCF-7 cells were cultured in 6-well plates at a density of 5×10⁵ per well. When cell confluence reached 70–80% (after culturing for 24 h), the culture medium was subsequently removed. On the next day, 20 nmol miR-143-5p mimics and NC mimics were mixed with 5 µl Lipofectamine^® 2000 in 500 µl OptiMEM (cat. no. 11668019; Invitrogen; Thermo Fisher Scientific, Inc.), incubated for 15 min with 37°C and then added to the culture medium. After 48 h, cells were harvested and subjected to the subsequent experiments. The miR-143-5p mimics and the NC mimics were obtained from Shanghai GenePharma Co., Ltd. The sequences were as follows: miR-143-5p mimics, 5′-GGUGCAGUGCUGCAUCUCUGGU-3′ and NC mimics, 5′-UGAGAUCAAGGAUUGAACCUC-3′, respectively. The HIF-1α agonist fenbendazole-d3 (cat. no. HY-B0413S; MedChemExpress LLC; 10 µmol/l) was used to activate the HIF-1α-related GLUT1 pathway. Briefly, when the cells were transfected with miR-143-5p mimics, the 20 µM HIF-1α agonist was also added to the culture medium. After incubation at 37°C for 48 h, the cells were harvested and subjected to the following experiments.

The proliferation of BT-549 and MCF-7 cells was analyzed using the Cell Counting Kit-8 (CCK-8) assay. Transfected cells (1×10³/well) were cultured in a 96-well plate with five replicates at 37°C with regulated humidity. Subsequently, 11 µl CCK-8 reagent (C0037; Beyotime Institute of Biotechnology) was added into each well at different periods (24, 48, 72 and 96 h) after transfection at 37°C. Following 4 h of culture, spectrometric absorbance at 450 nm was measured at 0, 12, 24, 48 and 72 h using a microplate reader.

Post-transfection, MCF-7 and BT-549 cells were cultured for 24 h. Subsequently, cells were cultured in a 6-well plate (2.5×10⁴ cells/well) for an additional 10 days. After this, During the incubation, transfections were renewed every 2 days. The the cells were then washed twice with phosphate-buffered saline (PBS) for 3 min, and the cell colonies were stained for 5 min with 0.5% crystal violet supplemented with 20% methanol at room temperature. Images were captured and colonies with >50 cells were selected and counted.

Using Transwell chambers, the ability of BT-549 and MCF-7 cells to migrate was measured. Briefly, cells (9×10⁴) were suspended in serum-free culture medium in a 24-well plate and then seeded into the upper chamber of a Transwell system (pore size, 8 µm; BD Biosciences). A total of 800 µl complete medium with 10% FBS was added to the lower chamber. Cells were incubated for 24 h at 37°C, followed by staining with 0.1% crystal violet for 30 min at room temperature. The cells migrating to the lower chamber were observed under an inverted microscope (Zeiss, CFM-500). Image Pro-Plus 6.0 software (Media Cybernetics) was used to assess migration.

To determine whether HIF-1α was a target gene of hsa-miR-143-5p, TargetScan version 7.2 online software (www.targetscan.org) was used. The HIF-1α 3′-UTR was synthesized and cloned into the pmirGLO plasmid (Shanghai GenePharma Co., Ltd.). Cells were transfected with 0.1 µg reporter plasmid pmirGLO containing wild type (WT) or mutated (MUT) HIF-1α, and 0.4 µg NC mimics or miR-143-5p mimics using Lipofectamine 2000 transfection reagent (cat. no. 11668019; Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h of transfection, luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity, and the relative luciferase activity is presented as a ratio of firefly luciferase intensity to Renilla luciferase intensity.

Total protein was extracted from BT-549 and MCF-7 cells using radioimmunoprecipitation assay lysis buffer (Bejing ComWin Biotech Co.) containing a protease inhibitor mixture. The BCA protein assay kit (Beyotime Institute of Biotechnology) was applied for determining protein concentration. Subsequently, equal amounts of protein (10 µg) were separated on 10% gels using SDS-PAGE and proteins were transferred to a nitrocellulose membrane (Amersham; Cytiva). After blocking with the 5% normal goat serum for 1 h at room temperature, the membranes were then incubated with β-actin (catalog no. ab8226; 1:10,000; Abcam), HIF-1α (catalog no. ab179483; 1:10,000; Abcam) and GLUT1 (catalog no. ab115730; 1:10,000; Abcam) antibodies at 4°C overnight, washed and further incubated with the goat anti-mouse IgG-HRP secondary antibodies (catalog no. BS12471; 1:5,000; Bioworld Technology, Inc.) for 1 h at room temperature. ECL (catalog no. P0018FS; Beyotime Institute of Biotechnology) was then used to visualize the blots and Alpha View software (version no. 3.2.2.0; ProteinSimple, Inc.) was used to analyze them. β-actin was used as a loading control.

TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) reagent was used to extract total cellular and tissue miRNA, followed by usage of the miRVana Isolation Kit (Ambion; Thermo Fisher Scientific, Inc.). RNA concentration was measured with a NanoDrop ND-2000 spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc.). After RT of miRNA to cDNA using the TaqMan MicroRNA Reverse Transcription tool (Applied Biosystems; Thermo Fisher Scientific, Inc.), the expression levels of miR-143-5p were assessed using the Prime Script miRNA RT-PCR Kit (Takara Bio, Inc.). A total volume of 10 µl was used for qPCR with the following thermocycling conditions: 95°C for 30 sec for initial denaturation, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Expression was determined using the 2^−ΔΔCq method (34). The primer sequences were as follows: miR-143-5p, forward 5-GGTGCAGTGCTGCATCT-3, reverse, 5-CTCAACTGGTGTCGTGGA-3; and U6, forward, 5-GCTTCGGCAGCACATATACTAAAAT-3, reverse 5-CGCTTCACGAATTTGCGTGTCAT-3.

BT-549 and MCF-7 cells (1×10⁴ cells) were added to culture dishes and then fixed with 4% paraformaldehyde for 20 min at room temperature. After incubating with 10% goat serum (catalog no. C0265; Beyotime Institute of Biotechnology) for 1 h at room temperature and 0.3% hydrogen peroxide for 30 min at room temperature, BT-549 and MCF-7 cells were incubated with primary antibodies against HIF-1α (catalog no. ab179483; 1:500; Abcam) and GLUT1 (catalog no. ab115730; 1:500; Abcam) antibodies overnight at 4°C. Cells were then washed three times with PBS plus 1% Tween 20, followed by a 45-min incubation with Alexa Fluor 488-labeled goat anti-rabbit IgG(H+L)(catalog no. C0265; Beyotime Institute of Biotechnology) and Alexa Fluor 555-labeled donkey anti-mouse IgG(H+L) (catalog no. A0460; Beyotime Institute of Biotechnology) at room temperature. The cell nuclei were subsequently stained with DAPI (Wuhan Boster Biological Technology, Ltd.) for 3 min at room temperature. The positive cells were observed under an inverted fluorescence microscope (Zeiss; YKDZ-80). Image Pro-Plus 6.0 software (Media Cybernetics, Inc.) was used to count the number of positive cells.

To explore the target gene of miR-143-5p, transcriptome sequencing analysis was performed in BC cells transfected with NC mimics or miR-143-5p mimics. Since miR-143-5p had similar biological effects on BT-549 and MCF-7 cells, one of the two cell lines was randomly selected for RNA sequencing. MCF-7 was used for RNA transcriptome sequencing. Briefly, a Small RNA Sample Prep Kit (catalog no. RS-200-0048; Illumina, Inc.) was used to construct miRNA libraries. A total of 3 ng RNA per sample was used as input material for the RNA sample preparations. In brief, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was performed using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (catalog no. SO142; Thermo Fisher Scientific, Inc.). Second strand cDNA synthesis was subsequently performed using DNA Polymerase RNase H. Remaining overhangs were converted into blunt ends using exonuclease/polymerase activities. After adenylation of the 3′ ends of the DNA fragments, the NEBNext Adaptor with a hairpin loop structure was ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150–200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Inc.). Next, 3 µl USER Enzyme (New England Biolabs, Inc.) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by 5 min at 95°C before PCR. PCR was performed using Phusion High-Fidelity DNA polymerase (catalog no. F630S; Thermo Fisher Scientific, Inc.), Universal PCR primers (catalog no. 48190011; Thermo Fisher Scientific, Inc.) and Index (X) Primer [catalog no. 12611ES02; Yeasen Biotechnology (Shanghai) Co., Ltd.]. Finally, the PCR products were purified (AMPure XP system), and the library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (catalog no. FC-401-3002; Illumina, Inc.), according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an IlluminaHiSeq 2000 platform and paired-end reads were generated. Differentially expressed mRNAs were analyzed by Limma packages in R version 4.1.2 software (https://www.r-project.org/).

The R language ‘limma’ package was used to analyze mRNA with differential expression based on the P<0.001 and |logFC|>2 thresholds. Subsequently, the pathway enrichment analysis was performed using OmicShare online platform (https://www.omicshare.com/) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG). Subsequently, protein interaction network diagrams were constructed and visualized with Cytoscape 3.7.2 software (http://cytoscape.org/).

Experiments were repeated three times. The data are presented as the mean ± SD and were analyzed using SPSS 23.0 software (SPSS, Inc.). Fisher's exact test was used to analyze the association between the miR-143-5p expression and clinicopathological features. The CCK-8 assay data were compared by two-way analysis of variance (ANOVA) followed by Bonferroni's correction. The difference between two groups was analyzed using unpaired Student's t-test, whereas one-way ANOVA followed by Bonferroni's correction was adopted to compare several groups. Survival rate was assessed using the Kaplan-Meier test and log-rank test. Pearson's correlation analysis was used to analyze the correlation of expression data. P<0.05 was considered to indicate a statistically significant difference.

After analysis of the GEO chip-based screening standards, i.e., |log FC|>2 and P<0.001, 40 differentially expressed miRNAs were identified between 28 BC tissues and 20 adjacent tissues from patients with BC in the GSE42072 dataset. miR-143-5p expression was increased within the GSE42072-derived BC samples compared with in the matched non-cancerous samples (P<0.001; Fig. 1A). In addition, there were 29 differentially expressed miRNAs between 32 BC pre-operative serum samples and 22 volunteer serum samples in the GSE41922 dataset. miR-143-5p expression was higher in BC pre-operative serum compared with that in volunteer serum (P<0.001; Fig. 1B).

BC and matched non-cancerous tissue samples were collected from 40 patients with BC; it was revealed that compared with those in paired adjacent tissues, the expression levels of miR-143-5p were significantly decreased in BC tissues (P<0.05; Fig. 1C). Kaplan-Meier survival analysis revealed that the survival rate of patients with high miR143-5p expression (split according to mean miR-143-5p expression levels) was higher than that in patients with lower expression levels of miR-143-5p (P<0.05; Fig. 1D and E). As shown in Table I, miR-143-5p expression was revealed to be associated with tumor size (P<0.05), N stage (P<0.05) and M stage (P<0.05), but not age (P>0.05), menopause (P>0.05), progesterone receptor status (P>0.05), human epidermal growth factor receptor status (P>0.05) or Ki67 levels (P>0.05). Moreover, miR-143-5p expression levels were significantly decreased in various BC cell lines compared with those in human common breast epithelial cells (P<0.05; Fig. 1F).

Since miR-143-5p expression levels were decreased in various BC cell lines, two cell lines were randomly selected to explore the biological role of miR-143-5p. Finally, miRNA mimics were used to upregulate the expression of miR-143-5p in BT-549 and MCF-7 cells. Post-transfection with miR-143-5p mimics, the expression levels of miR-143-5p were significantly increased compared with in cells transfected with NC mimics (Fig. 2A). Subsequently, the role of miR-145-5p in cell proliferation was determined by CCK-8 assay; notably, overexpression of miR-143-5p reduced the proliferative potential of BT-549 (P<0.05; Fig. 2B) and MCF-7 (P<0.05; Fig. 2C) cells. When compared with the NC mimics group, overexpression of miR-143-5p significantly decreased the colony-forming capacity of BC cells (P<0.05; Fig. 2D and E). Moreover, as determined using the Transwell assay, overexpression of miR-143-5p suppressed the migration of BC cells (P<0.05; Fig. 2F and G).

To study the detailed mechanism by which miR-143-5p inhibited the tumorigenesis of BC cells, differentially expressed genes were analyzed using the R language ‘limma’ package. The results showed that 34 differentially expressed genes were identified in BC cells transfected with miR-143-5p mimics (Fig. 3A and B). Subsequently, the pathway enrichment analysis of differentially expressed genes was carried out, and the HIF-1α-related pathway was identified (Fig. 3C). The PPI network of the differentially expressed genes is shown in Fig. 3D, which suggests that HIF-1α acts on GLUT1.

To predict whether HIF-1α is a potential target gene of miR-143-5p, TargetScan 7.2 was used; it was revealed that miR-143-5p targets HIF-1α (Fig. 4A). According to the dual-luciferase reporter assay, miR-143-5p significantly decreased luciferase activity when cells were co-transfected with the reporter containing WT HIF-1α 3′-UTR compared with the reporter containing MUT HIF-1α 3′-UTR, which further indicated that miR-143-5p can target HIF-1α (P<0.05; Fig. 4B and C). After transfection with the miR-143-5p mimics, the protein expression levels of HIF-1α and GLUT1 were decreased in BT-549 (P<0.05, Fig. 4D and E) and MCF-7 cells (P<0.05; Fig. 4D and F). There was a significant inverse correlation between miR-143-5p and HIF-1α protein expression (P<0.05; Fig. 4G and J), and between miR-143-5p and GLUT1 protein expression (P<0.05; Fig. 4H and K). There was a positive correlation between HIF-1α and GLUT1 protein expression in BT-549 (P<0.05; Fig. 4I) and MCF-7 cells (P<0.05; Fig. 4L).

In the immunofluorescence assay, green fluorescence indicated that HIF-1α (P<0.05) and GLUT1 (P<0.05) were significantly lower in the miR-143-5p mimics group compared with in the NC mimics group (Fig. 5A, B, F and G). Additionally, there was a negative correlation between miR-143-5p expression and HIF-1α (P<0.05; Fig. 5C and H), and between miR-143-5p expression and GLUT1 (Fig. 5D and I). Moreover, HIF-1α fluorescence expression was positively correlated with that of GLUT1 in BT-549 (P<0.05; Fig. 5E) and MCF-7 (P<0.05; Fig. 5J) cells.

To explore the role of miR-143-5p in suppressing BC cell carcinogenesis via the HIF-1α-related GLUT1 pathway, HIF-1α agonist fenbendazole-d3 was used to activate the HIF-1α-related GLUT1 pathway. Notably, the protein expression levels of HIF-1α and GLUT1 were elevated by fenbendazole-d3 in BT-549 (P<0.05; Fig. 6A and B) and MCF-7 cells (P<0.05; Fig. 6A and C). In addition, the inhibiting influence exerted by miR-143-5p on proliferation (P<0.05; Fig. 6D and E), colony-forming capacity (P<0.05; Fig. 6F and G), and migration (P<0.05; Fig. 6H and I) was reversed by the HIF-1α agonist.