The University of Alabama at Birmingham Cancer data analysis portal (UALCAN; http://ualcan.path.uab.edu/analysis.html) was used to analyze the expression levels of IRX5 and YWHAB in different subtypes of breast cancer cells in The Cancer Genome Atlas (TCGA) database. The dataset used was TCGA dataset: Breast invasive carcinoma in UALCAN. Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cancer-pku.cn/index.html) was used to analyze the correlation between YWHAB and IRX5 expression in breast cancer. The dataset used was breast invasive carcinoma (BRCA) tumor (BRCA: breast cancer) in GEPIA.

MDA-MB-231, MCF7 and 293FT cell lines were purchased from Procell Life Science & Technology Co. Ltd. The MDA-MB-231 cells were cultured in DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (MilliporeSigma), 100 U/ml penicillin and 100 µg/ml streptomycin. The MCF7 and 293FT cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). All cells were cultured in a saturated humidity incubator containing 5% CO₂ at 37°C.

The promoter sequence upstream of the human YWHAB transcription start site (NC_000020.11: 44883702-44885654) was obtained by PCR amplification. 293FT cell genomic DNA as the PCR template was extracted using the Genomic DNA Mini-Preps Kit (Beijing Baiolaibo Technology Co., Ltd.). Prime STAR GXL DNA Polymerase, dNTP Mixture and 5X PrimeSTAR GXL Buffer were purchased from Takara Biotechnology Co., Ltd. The primers used are shown in Table I. The PCR reaction procedure was as follows: 98°C for 2 min, followed by 30 cycles of 98°C for 10 sec, 50°C for 15 sec and 68°C for 2 min; and final extension at 68°C for 10 min. PCR products were detected using a 1% agarose gel containing ethidium bromide and bands were observed using a gel imaging system (ProteinSimple). Finally, the correctness of the sequence was verified by Sanger sequencing (Sangon Biotech Co., Ltd.). The YWHAB-promoter-PGL3 vector was obtained by ligating the promoter sequence into the pGL3-basic vector (Promega Corporation). The open reading frames of the IRX5 (NM_005853.6) gene were cloned into a pcDNA3.1(+) vector (Invitrogen; Thermo Fisher Scientific, Inc.). For the luciferase assay, 5×10⁴ 293FT cells were plated and cultured in 24-well plates overnight at 37°C until they reached 80% confluence, after which, they were co-transfected with YWHAB-promoter-PGL3 vector, pcDNA3.1-IRX5 vector and PGMR-TK Renilla luciferase reporter plasmid (Genomeditech) using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). Firefly and Renilla luciferase activity were measured after transfection for 24 h using a Dual Luciferase Reporter Gene Assay Kit (Beyotime Institute of Biotechnology).

The coding sequences (CDSs) of the YWHAB gene (GenBank NM_139323.4) were obtained by reverse transcription-PCR (RT-PCR) and were checked by Sanger sequencing (Sangon Biotech Co., Ltd.). RNAiso Plus reagent (Takara Biotechnology Co., Ltd.) was used to extracted RNA from 293FT cells, and the PrimeScriptTMRT reagent Kit with gDNA Eraser (Takara Biotechnology Co., Ltd.) was used to reverse transcribe RNA into cDNA. The primers used are shown in Table I. The PCR reaction procedure used was as follows: 98°C for 2 min; 98°C for 30 sec, 50°C for 30 sec and 68°C for 1 min for 30 cycles; 68°C for 1 min. The verified CDSs were ligated into the lentiviral vector PEB-3XFlag-GP (Guangzhou Huijun Biotechnology Co., Ltd.) and were packaged using the GM easy™ lentiviral Packaging Kit (Genomeditech; third-generation lentiviral packaging system). The lentiviral transduction plasmid:packaging plasmid:envelope plasmid ratio was 2:1:1. Briefly, 10 µg of the aforementioned recombinant plasmid and 10 µg of the GM easy™ Lentiviral Mix plasmid (Genomeditech) were co-transfected into 293FT cells (10-cm Petri dish; 80% confluence) using 60 µl HG Transgene™ Reagent (Genomeditech). The cells were cultured at 37°C with 5% CO₂ for 18 h and then the culture medium was replaced. After culturing for 48 h, the supernatant was collected and contained the lentiviral particles. The lentiviral particles (MOI, 20) were added to MDA-MB-231 cells and the culture medium was replaced 12 h later. Empty PEB-3XFlag-GP was packaged and transduced simultaneously as a control. After 48 h, puromycin (2 µg/ml) was used for selection for 2 weeks and 0.5 µg/ml puromycin was used for maintenance. The time interval between transfection and subsequent experimentation was 14 days. Western blotting and RT-quantitative PCR (RT-qPCR) were used to detect the YWHAB transduction efficiency. MDA-MB-231 cells were transduced with the empty lentiviral vector or YWHAB-linked lentiviral vector and were named MDA-MB-231-VC and MDA-MB-231-YWHAB, respectively. The lentiviral vector used to knock down YWHAB was purchased from Guangzhou IGE Biotechnology Co., Ltd. The short hairpin RNA (sh) sequence targeting YWHAB and the scrambled negative control are shown in Table I. Lentiviral packaging and transduction were performed in the aforementioned manner. The MCF7 cells with knockdown of YWHAB were referred to as MCF7-shYWHAB cells, and the control cells were referred to as MCF7-CT cells.

Total RNA was extracted from MDA-MB-231, MCF7 and 293FT cells using RNAiso Plus reagent (Takara Biotech Co., Ltd.) and was reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. qPCR was performed using the 2X SG Fast qPCR Master Mix Kit (Sangon Biotech Co., Ltd.) on a LightCycler96 (Roche Diagnostics) with the following thermocycling conditions: Initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 3 sec, 60°C for 30 sec and 97°C for 1 sec. mRNA levels were quantified using the 2^−ΔΔCq method and normalized to the internal reference gene GAPDH (20). The primers used in the present study are shown in Table I.

Proteins were extracted from cells (MDA-MB-231-VC, MDA-MB-231-YWHAB, MCF7-CT and MCF7-shYWHAB cells) using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology) and the concentration was then adjusted to 4 µg/µl. The proteins (60 µg/lane) were separated by SDS-PAGE on a 10% gel and then transferred onto PVDF membranes (MilliporeSigma) for 90 min at 4°C at 150 mAh, and the membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with the following primary antibodies overnight at 4°C: Mouse anti-Flag (1:1,000; cat. no. D191041; Sangon Biotech Co., Ltd.), rabbit anti-vimentin (1:1,000; cat. no. 5741T; Cell Signaling Technology, Inc.), rabbit anti-YWHAB (1:1,000; cat. no. BM4752; Boster Biological Technology) and mouse anti-GAPDH (1:1,000; cat. no. D190090; Sangon Biotech Co., Ltd.). The membranes were then washed three times with TBS with 0.1% Tween-20 and incubated with the following HRP-conjugated secondary antibodies at 4°C for 2 h: HRP-conjugated Goat Anti-Mouse (1:5,000; cat. no. D110087; Sangon Biotech Co., Ltd.) and HRP-conjugated Goat Anti-Rabbit (1:5,000; cat. no. D110058; Sangon Biotech Co., Ltd.). The antibody against the Flag tag was used to examine the exogenous YWHAB protein with the Flag tag. Finally, an HRP-DAB color development kit (Tiangen Biotech Co., Ltd.) or ultrasensitive luminescence reagent (Sangon Biotech Co., Ltd.) was used for visualization. Images were captured using an image analysis system (5200; Tanon Science and Technology Co., Ltd.) and protein bands were semi-quantified using ImageJ 1.8 (National Institutes of Health).

The MTT assay was performed to examine cell proliferation. MDA-MB-231-VC and MDA-MB-231-YWHAB cells were seeded at a density of 5×10³ cells/well in 96-well plates and cultured for 1, 2, 3, 4 and 5 days. The cells were then incubated with MTT reagent (Beijing Solarbio Science & Technology Co., Ltd.) for 4 h. DMSO (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well to dissolve formazan crystals, and the absorbance was determined at 490 nm using a microplate reader (Super Max 3100; Shanghai Shanpu Biotechnology Co., Ltd.).

Cell colony formation was measured using a plate colony formation assay. MDA-MB-231-VC and MDA-MB-231-YWHAB cells were seeded into 6-well plates at a density of 200 cells/well and were cultured at 37°C in 5% CO₂ for 2 weeks until visible cell colonies were formed. Cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Before counting, the cell colonies were gently washed with PBS and then stained with 0.1% crystal violet for 15 min at room temperature. Viable colonies containing ≥50 cells were counted manually using an inverted light microscope (DP72; Olympus Corporation).

To assess breast cancer cell migration, a wound healing assay was performed. MDA-MB-231-VC and MDA-MB-231-YWHAB cells were seeded in 6-well plates at a density of 2×10⁵ cells/well and were cultured at 37°C for 24 h. When the cells achieved stable attachment and reached 90% confluence, a parallel linear scratch was generated using a sterile 200-µl pipette tip. After rinsing with PBS, the wounded cells were cultured in serum-free DMEM (Gibco; Thermo Fisher Scientific, Inc.). Images were captured using an inverted light microscope (DP72; Olympus Corporation) after 0, 24 and 48 h of incubation at 37°C. The areas were measured using ImageJ 1.8 (National Institutes of Health). The cell wound healing rate can reflect migration, and was calculated as follows: Relative migration rate (%)=closure area/initial area ×100%.

Cell invasion and migration were assessed using 24-well Transwell plates (8-µm pore size; Corning, Inc.) and a Cell Invasion Chamber (8-µm pore size; Beijing Labselect). The pre-coating with Matrigel of the Cell Invasion Chamber was completed by the manufacturer. Cells (5×10⁴; MDA-MB-231-VC, MDA-MB-231-YWHAB, MCF7-CT and MCF7-shYWHAB cells) were collected and seeded into the upper chambers in serum-free culture medium. Medium containing 10% FBS was added to the lower chambers to attract cells migrating and invading through the membranes. After incubation at 37°C for 24 h, the cells in the upper chambers were removed by washing with PBS. The cells that had migrated and invaded through the membranes of the chambers were fixed with paraformaldehyde at room temperature for 15 min, stained with crystal violet at room temperature for 15 min and counted under an inverted light microscope (DP72; Olympus Corporation).

The transcriptome analysis of MDA-MB-231-YWHAB and MDA-MB-231-VC cells was performed by Novogene Co., Ltd. Total RNA was isolated from cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Total RNA was identified and quantified as follows: 1% agarose gel electrophoresis was used to analyze the degree of RNA degradation and DNA contamination in the sample; a spectrophotometer (NanoPhotometer; Implen GmbH) was used to measure RNA purity and concentration; RNA integrity and quantity were measured using the RNA Nano 6000 Assay Kit (Agilent Technologies, Inc.) on the Bioanalyzer 2100 system (Agilent Technologies, Inc.). The kits used to Library preparation included: ABclonal First Strand synthesis module (cat. no. RK20353L; ABclonal Biotech Co., Ltd.), ABclonal Second Strand synthesis module (cat. no. RK20346L; ABclonal Biotech Co., Ltd.) and KC-Digital Stranded mRNA Library Prep Kit for Illumina-UMI (cat. no. DR085-01/02; Seqhealth). Library quantification was performed using a Qubit2.0 fluorometer (Invitrogen; Thermo Fisher Scientific, Inc.). Libraries were sequenced with a NovaSeq 6000 S4 Reagent Kit v1.5 (300 cycles; (cat. no. 20028312; Illumina, Inc.). The final library loading concentration was 5 nM. An Illumina Novaseq 6000 (Illumina, Inc.) was used to conduct paired-end sequencing with the PE150 mode according to the manufacturer's protocol. UMI-tools (v1.1.2) was used to identify the unique molecular identifier sequence on each sequence in the clean reads (21). Cleaning of reads was performed by deleting reads that contain adapter reads, plot-N reads and low-quality reads. HISAT2 v2.1.0 was used to construct a reference genome index and perform reference genome alignment on paired-end clean reads (22). StringTie (v2.2.1) was used for new gene prediction (23). FeatureCounts (v2.0.1) was used to count reads mapped to each gene (24). Differential expression analysis of the two cell groups was performed using the edgeR software package (v3.22.5) (25). The genes with a log₂ fold change value >1 and P<0.05 were considered significantly differentially expressed genes (DEGs). GO (http://geneontology.org/) and KEGG (https://www.genome.jp/kegg/) enrichment analyses were performed to evaluate the biological function of YWHAB and to investigate the pathways in which the DEGs were involved. GO and KEGG enrichment analysis was performed using the clusterProfiler R package (3.8.1) (26).

All data were processed using the statistical software SPSS 20.0 (IBM Corp.) and GraphPad Prism 8.0 (Dotmatics). The data are presented as the mean ± SD. Differences between means were evaluated using unpaired Student's t-tests or one-way analysis of variance with Tukey's post hoc test. All experiments were repeated three times. P<0.05 was considered to indicate a statistically significant difference.

Using TCGA, it was demonstrated that the expression levels of IRX5 and YWHAB were both decreased with the increase in the metastatic ability of breast cancer cell subtypes (metastatic ability of breast cancer cell subtypes: Luminal < HER2-positive < triple-negative) (Fig. 1A). This showed that the higher the metastatic ability of breast cancer subtypes, the lower the expression levels of YWHAB and IRX5. High expression of YWHAB may inhibit the metastatic ability of breast cancer. The very weak positive association between YWHAB and IRX5 gene expression was determined by Pearson correlation analysis using GEPIA (Fig. 1B). The correctness of the YWHAB promoter sequences was verified using Sanger sequencing (Fig. 1C). To determine whether IRX5 interacts with the YWHAB promoter, a dual-luciferase assay was used to detect the interaction between IRX5 and the YWHAB promoter. Compared with PCDNA3.1-IRX5 or PGL3-YWHAB-promoter alone, the simultaneous addition of both significantly increased the luciferase activity (Fig. 1D). This indicated that IRX5 significantly increased luciferase activity by affecting the YWHAB promoter. 293FT cells were transfected with pcDNA3.1-IRX5 plasmid at numerous concentrations to confirm the regulatory effect on the promoter. As the concentration of pcDNA3.1-IRX5 plasmid increased, the luciferase activity also increased (Fig. 1E). These results suggested that IRX5 may regulate YWHAB gene expression by affecting the YWHAB promoter.

To elucidate the function of YWHAB in breast cancer cells, YWHAB was overexpressed in MDA-MB-231 cells. Sanger sequencing was performed to verify the correctness of YWHAB coding sequences (Fig. 2A). The overexpression of YWHAB was verified by RT-qPCR and western blotting. YWHAB mRNA expression was significantly increased in the cells transfected with the YWHAB overexpression plasmid compared with those transfected with the empty vector (Fig. 2B). Western blotting also demonstrated successful transduction using the Flag antibody to detect Flag-tagged YWHAB protein (Fig. 2C). The MTT assay was performed to identify the effect of YWHAB overexpression on cell proliferation. Cell proliferation curves demonstrated that there was no significant difference in the proliferation of cells between the YWHAB-OE and VC groups (Fig. 2D). No significant difference in colony numbers was observed between the YWHAB-OE and VC groups (Fig. 2E). These findings indicated that YWHAB overexpression may have no effect on breast cancer cell proliferation.

The characteristics of cancer cells also include migration and invasion. The aforementioned results demonstrated that YWHAB had no significant effect on cell proliferation. The effect of overexpression of YWHAB on cell migration and invasion was assessed using wound healing and Transwell assays. In the wound healing assay, the relative migration rate of the YWHAB-OE group was significantly reduced at 48 h compared with that of the VC group (Fig. 3A). The Transwell assay demonstrated that YWHAB-OE significantly reduced cell migration and invasion compared with that of the VC group (Fig. 3B and C). Furthermore, YWHAB expression was knocked down in MCF7 breast cancer cells, which was verified by RT-qPCR and western blotting. The results demonstrated that in MCF7-shYWHAB cells, the mRNA levels of YWHAB were significantly reduced compared with those in the control group (Fig. 4A). Western blotting demonstrated a notable decrease in the protein levels of YWHAB in MCF7-shYWHAB cells (Fig. 4B). Furthermore, the Transwell assays demonstrated that YWHAB knockdown significantly increased cell migration and invasion compared with those in the control group (Fig. 4C and D). These results indicated that YWHAB could inhibit cell migration in vitro, which suggests that YWHAB may act as a tumor suppressor gene.

To clarify the molecular pathways that are involved in YWHAB-induced suppression of cell migration and invasion, transcriptome analysis was performed in YWHAB-overexpressing cells and control cells. The data from this assay have been uploaded to the Gene Expression Omnibus database (GSE218458). A total of 61 genes were demonstrated to be differentially expressed, among which 43 genes were upregulated and 18 genes were downregulated when YWHAB was overexpressed in MDA-MB-231 cells (Fig. 5A). GO and KEGG pathway enrichment analyses were used to assess the pathways and molecular functions in which the DEGs were involved, in order to better understand the functions of YWHAB overexpression. GO was used to classify gene functions where each DEG could be assigned to one or more GO terms within three domains. ‘Response to stimulus’ (GO:0050896), ‘cellular response to stimulus’ (GO:0051716) and ‘localization’ (GO:0051179) were enriched in the biological process domain. Within the cellular component domain, the three most enriched categories were ‘extracellular region’ (GO:0005576), ‘extracellular region part’ (GO:0044421) and ‘vesicle’ (GO:0031982). In terms of molecular functions, the enriched categories were ‘cytokine activity’ (GO:0005125), ‘receptor binding’ (GO:0005102) and ‘extracellular matrix structural constituent’ (GO:0005201) (Fig. 5B). The DEGs were mainly associated with the terms relating to the extracellular region, environmental stimuli and binding to receptors, suggesting that YWHAB may be involved in these signaling pathways affecting metastasis of cells. The DEGs were also assigned to the biological pathways described in KEGG to better understand their specific metabolic pathways in cells (Fig. 5C). The ‘TNF signaling pathway’ (KEGG: map04688) is related to cancer processes. The pathway with the largest number of DEGs was ‘Rheumatoid arthritis’ (KEGG: map05323). These results indicated that YWHAB and DEGs may affect the metastasis of breast cancer cells by participating in multiple signaling pathways.

By searching existing studies, three genes (MMP1, MMP3 and IL24) related to breast cancer or EMT were selected (27–32). In the present study, both transcriptome analysis results and RT-qPCR detection indicated that MMP1, MMP3 and IL24 were downregulated (Fig. 6A). In addition, five DEGs (FAM20C, PLEC, TNFSF15, DDIT3 and HERPUD1) were randomly selected for RT-qPCR detection to verify whether their expression changes were consistent with the transcriptome analysis results. The results showed that FAM20C and PLEC were upregulated in MDA-MB-231-YWHAB cells. TNFSF15, DDIT3 and HERPUD1 were downregulated (Fig. 6A). The upregulation and downregulation trends of these genes detected by RT-qPCR were consistent with the trends in transcriptome analysis.

Vimentin is a mesenchymal marker that is associated with EMT (33). The changes in vimentin expression after overexpression and knockdown of YWHAB were assessed. After the overexpression of YWHAB, the protein levels of vimentin were decreased, whereas after the knockdown of YWHAB, the protein levels of vimentin were increased (Fig. 6B). The aforementioned results suggest that there may be an association between YWHAB and vimentin.