A total of 50 pairs of BC and adjacent normal (N) tissues were collected from Hubei Cancer Hospital (Wuhan, China) between December 2019 and May 2020. The patients with BC were aged between 55 and 65 years, and received no drug therapy before tumor removal. Immediately following surgery, all the tissues were frozen in liquid nitrogen and maintained at −80°C until further analysis. All the clinical samples were collected with written informed consent from the patients, and the protocol was approved by the Ethics Committee of Hubei Cancer Hospital (approval no. 2021-IEC213).

The breast MCF10A epithelial cell line, the BC cell lines (MDA231, MCF7, MDA468 and BT474) and 293T cell line were purchased from BeNa Culture Collection (Beijing, China). The cell lines were free from mycoplasma contamination. The MDA231, MCF7, MDA468 and BT474 cell lines were cultured in DMEM supplemented with 10% FBS and 100 U/ml penicillin and streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.). The MCF10A cell line was maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin (ScienCell Research Laboratories, Inc.). The cell lines were maintained at 37°C in a humidified atmosphere with 5% CO₂. In addition, hypoxia treatment comprised 94% N₂, 5% CO₂ and 1% O₂ at 37°C for different durations under hypoxic conditions. Hypoxia was simulated by culturing cells with CoCl₂ (Sigma-Aldrich) at a final concentration of 100 µM.

The short hairpin (sh)RNAs targeting MBP1 and the corresponding negative control (NC) shRNA were constructed by TSINGKE (Tianjin, China) and were transfected with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at a final concentration of 50 ng and 37°C for 48 h. Transient transfection was confirmed using reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis. The following shRNA MBP1sequences were used: MBP1 sense, 5′-GCUGCUUACUGUAACUGUAUC-3′ and antisense, 5′-UACAGUUACAGUAAGCAGCUG-3′; NC shRNA sense, 5′-AAAAATTCAAGACUUGGAGCU-3′ and antisense, 5′-UCUUGTTUUUUUAGCUCCAAG-3′. MBP1 lentivirus (Lv-MBP1) and empty control vector (Lv-NC) were constructed by Shanghai GeneChem Co., Ltd., according to the shRNA sequences of MBP1 based on 3rd generation system. Recombinant lentiviruses were amplified (Plasmid transfection concentration: 20 µg/1×10⁷ cells) in 293T cells and purified by centrifugation and subsequent analysis. The recombinant lentiviruses were stably infected into the BC cell lines (MCF7 and MDA231) at a multiplicity of 2 and 37°C for 48 h. Cells were used for subsequent experiments after 3 days of puromycin selection at a final concentration of 2 µg/ml. All the experiments were repeated independently at least three times.

The BC cells were counted using the CCK-8 assay according to the manufacturer's instructions to determine the proliferative ability of the cells. The BC cells were seeded in 96-well plates and incubated at 37°C for 24 h. Next, CCK-8 solution (10 µl) was added to each well and the cells were incubated for 2 h. Finally, the absorbance at 450 nm was analyzed. The experiment was replicated independently three times.

Colony formation assays were performed as previously described (18). All the colony formation assays were conducted in triplicate.

RT-qPCR was performed as previously described (18). The following primers were used: MBP1 forward, 5′-GGCGGTGACAGACTCCAAG-3′ and reverse, 5′-GAAGCTCGTCGGACTCTGAG-3′; β-catenin forward, 5′-AAAGCGGCTGTTAGTCACTGG-3′ and reverse, 5′-CGAGTCATTGCATACTGTCCAT-3′; and β-actin forward 5′-CCAAGGCCAACCGCGAGAAGATGAC-3′ and reverse 5′-AGGGTACATGGTGGTGCCGCCAGAC-3′. The experiments were performed in triplicate using a Bio-Rad CFX96 thermocycler (Bio-Rad Laboratories, Inc.), and the relative expression values were calculated using the 2^−∆∆Cq method (19).

Western blot analysis was performed as previously described (18). Briefly, protein lysate from MCF7 and MDA231 cells was extracted using RIPA buffer (Sigma-Aldrich; Merck KGaA) containing 1% PMSF and phosphatase inhibitors. The protein concentration of each sample was determined by a BCA protein assay kit (Beyotime, Shanghai, China). Denatured protein samples (10 µg/lane) were separated by 8% SDS-PAGE and trans-printed onto nitrocellulose filter (NC) membranes. The NC membranes were blocked with 5% non-fat dried milk in Tris-buffered saline (pH 7.4) containing 0.1% Tween-20 (TBST) for 1 h at room temperature, and subsequently reacted with the specific primary antibody in TBST at 4°C overnight. The membranes were washed for three times in TBST, 10 min each, and were incubated with a HRP-conjugated affinipure goat anti-rabbit IgG (H + L) (Proteintech, SA00001-2, Wuhan, China, 1:4,000 dilution) for 1 h at room temperature. Signal detection was developed using an enhanced chemiluminescence reaction (Meilunbio, Dalian, China).

IHC was performed as previously described (18). Briefly, the obtained tissue was incubated in 10% neutral formalin for 72 h at room temperature, paraffin-embedded tumor tissues were serially sectioned at 5-µm-thick sections. Serial tissue sections were antigen retrieved using 10 mM Sodium Citrate at 98°C for 20 min and were washed twice with 100% ethanol and 95% ethanol for 10 min each. Then, tissue sections were deparaffinized, blocked using 0.01 M citric acid buffer (pH 6.0) at 95°C for 15 min and incubated overnight at 4°C with primary antibodies followed by incubation for HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (Proteintech, SA00001-2, Wuhan, China, 1:5,000 dilution) for 1 h at room temperature. Then nuclei were counterstained using hematoxylin for 2 min at room temperature. The sections were then examined using a light microscope. The IHC results were scored by two independent observers according to both the percentage of positively stained cells (0, 1-25% staining; 1, 26-50% staining; 2, 51-75% staining; 3, 76-100% staining) and the staining intensity (scored from 0 to 3), and the final immunoreactivity score (IRS, range 0-9) was obtained by multiplying the two scores. The expression levels were classified as low if the score was less than 5 and as high if the score was 5 or higher.

The following antibodies were used: MBP1 (cat. no. 24207-1-AP; 1:1,000 dilution for western blot, 1:200 dilution for IHC), β-catenin (cat. no. 17565-1-AP; 1:1,000 dilution for western blot), HIF-1α (20960-1-AP; 1:1,000 dilution for western blot) and GAPDH (cat. no. 10494-1-AP; 1:2,000 dilution for western blot) (all from ProteinTech Group, Inc.). All the experiments were repeated independently at least three times.

The MCF7 cells (3×10⁶/mouse; >3/group) were transfected with Lv-MBP1 or Lv-NC and subcutaneously injected into the right flank of male BALB/c nude mice (6 weeks old; weight, ~15 g; 10 in total). All the nude mice were kept in a specific pathogen-free environment with controllable light (12-h light/dark cycle), temperature 18-29°C and relative humidity (40-70%) with food and water available ad libitum. The mice were monitored weekly, and the tumor volume was assessed; the long diameter of the tumor did not exceed 2 cm. The following formula was used to calculate the tumor volume: Volume (V)=LxW² × π/6, where L is the long diameter of the tumor and W is the short diameter of the tumor. Approximately 3 weeks after injection, the xenograft tumors had grown to a suitable size and met ethical requirements, according to institutional ethical guidelines. Then, the mice (9 mice) were anaesthetized with isoflurane (induction dose, 3-4% and maintenance dose, 1-1.5%) and sacrificed by humane cervical dislocation. Death was determined by respiratory arrest and the absence of chest fluctuations. The weights of the tumors were then recorded. Notably, one mouse was not included due to insufficient cell number during subcutaneous injection. The mouse experiments and handling of the animals were performed according to the Institutional and Animal Care and Use Committee of Hubei Cancer Hospital (approval no. 2019252A) and the NIH Guide for the Care and Use of Laboratory Animals.

Co-IP assay was performed using Co-IP kit (Abs955, Absin, Shanghai, China) according to the manufacturer's protocol. Briefly, MCF7 or MDA231 cells were homogenized in IP lysis buffer (20 mM Tris-HCl pH 7.5, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM DTT, 1 mM cocktail, 1 mM phosphoSTOP, 1 mM NEM, 1 mM NAM). A total of 500 µg extracts were incubated with indicated primary antibody or IgG as negative control for 4 h and protein A/G-Sepharose beads for 2 h at 4°C. The following antibodies were used: MBP1 (cat. no. 24207-1-AP; 1:200 dilution) and HIF-1α (cat. no. 20960-1-AP; 1:200 dilution) (both from ProteinTech Group, Inc.).

The β-catenin promoter region (2-kb sequence upstream of the transcription initiation site) and mutant (MUT) promoter were constructed into pGL3-based vectors. The BC cells were transfected with MBP1-sh and Lv-MBP1 along with pGL3 β-catenin using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at a final concentration of 50 ng. Firefly luciferase activity was measured 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega Corporation) and normalized to Renilla luciferase activity. The reporter plasmids were constructed by TSINGKE (Tianjin, China). All the experiments were repeated at least three times independently.

All statistical analyses were performed using SPSS v22.0 (IBM Corp.) software, and the figures were produced using GraphPad Prism v6.0 (GraphPad Software, Inc.). The data were presented as the mean ± SD, and differences between groups were analyzed using either an unpaired Student's t-test or one-way ANOVA followed by Tukey's post hoc test for experimental results. The median value of RT-qPCR results was used as a cutoff value for further analysis. Survival analysis was performed using the Kaplan-Meier method and log-rank test. Univariate analyses were performed using a χ² test. Online website (kmplot.com/analysis/) and data from TCGA were used for survival analysis. The Human Protein Atlas (proteinatlas.org) was used for analysis of MBP1 protein expression. P<0.05 was considered to indicate a statistically significant difference.

To analyze the expression patterns of MBP1 in BC, RT-qPCR was used to detect the mRNA expression level of MBP1 and the results, from 50 clinical samples, indicated that the mRNA expression levels of MBP1 were significantly reduced in BC tissues compared with that in paired N tissues (Fig. 1A). The protein expression level of MBP1 was detected using IHC and there was lower expression in BC tissues compared with that in paired N tissues (Fig. 1B). To further verify the expression results of MBP1 in clinical samples, RT-qPCR and western blot analysis was used to determine the mRNA and protein expression levels of MBP1 in the human breast epithelial cell line MCF10A and four BC cell lines (MDA231, MCF7, MDA468 and BT474), respectively. The mRNA and protein expression level of MBP1 in the four BC cell lines was lower compared with that in the normal human breast epithelial cell lines (Fig. 1C and D). Lastly, online analysis from Human Protein Atlas (proteinatlas.org) online database revealed that MBP1 protein expression in BC was low compared with that in normal breast epithelial cells (Fig. 1E). These results uniformly demonstrate that MBP1 expression was reduced in BC; therefore, MBP1 may be a tumor suppressor in BC.

Subsequently, the clinical relevance of MBP1 expression in patients with BC was analyzed. The MBP1 expression levels were divided into low and high expression groups according to the RT-qPCR and IHC results (Fig. 2A and B). The association between MBP1 expression levels and clinicopathological features was assessed in patients with BC (Table I). Univariate analysis showed that low MBP1 expression was positively associated with poor patient outcomes, including tumor size (P=0.003), histological grade (P=0.006), TNM stage (P=0.004), tumor stage (P=0.025), lymph node metastasis status (P=0.013), and metastasis status (P<0.001). Analysis of prognosis using Kaplan-Meier survival curves revealed that patients in the MBP1 high expression group had improved prognosis (P=0.028) (Fig. 2C). Further analysis of TCGA data (kmplot.com/analysis/) showed that the overall survival (OS) and disease-free survival (DFS) times were also improved in patients in the high MBP1 expression group (Fig. 2D and E). In addition, based on TCGA data, the prognosis of MBP1 was improved when endocrine therapy was administered to patients in the low MBP1 expression group (Fig. 2F). Therefore, MBP1 may be used as a clinical molecular indicator for endocrine therapy.

To further elucidate the role of MBP1 in BC tumor growth in vivo, MCF7 cells stably transfected with Lv-NC or Lv-MBP1 were injected into nude mice as xenografts (Fig. 3A). Western blot analysis indicated that MBP1 protein expression was high in the Lv-MBP1 group (Fig. 3B). Furthermore, MBP1 was also highly expressed from IHC and RT-qPCR (Fig. 3C and D). Notably, there was a significant reduction in tumor growth and final tumor weight in xenografts from mice in the Lv-MBP1 overexpression group (Fig. 3E and F). These results indicated that high MBP1 expression inhibits the proliferation of BC cells in vivo.

To investigate the possible mechanisms by which MBP1 inhibits the proliferation of BC, it was investigated whether MBP1 inhibited the proliferation of the BC cells. RT-qPCR and western blot analysis showed that Lv-MBP1 transfection notably increased the mRNA and protein expression level of MBP1 in the MDA231 and MCF7 cell lines compared with that in cells transfected with Lv-NC, respectively (Fig. 4A and B). Furthermore, the CCK-8 assay revealed that the MDA231 and MCF7 cells transfected with Lv-MBP1 had significantly inhibited proliferation compared with that in cells transfected with Lv-NC (Fig. 4C and D). In addition, the results from the colony formation assay revealed significantly inhibited colony formation in the MDA231 and MCF7 cell lines transfected with Lv-MBP1 compared with that in cells transfected with Lv-NC (Fig. 4E and F). By contrast, the MBP1-sh plasmid notably decreased MBP1 mRNA and protein expression levels in the MDA231 and MCF7 cell lines (Fig. 4G and H). Furthermore, MBP1-sh transfection significantly increased proliferation in the MDA231 and MCF7 cells (Fig. 4I and J), and low MBP1 expression increased colony formation in the MDA231 and MCF7 cells (Fig. 4K and L). Therefore, MBP1 may be a gene that inhibits the proliferation of BC cells.

β-catenin is a key molecule of the Wnt signaling pathway and a key marker to promote tumor proliferation (20). Therefore, it was investigated whether MBP1 regulated β-catenin expression. Lv-MBP1 overexpression in the MDA231 cell line notably decreased the mRNA and protein levels of β-catenin (Fig. 5A and B). Similar results were found in the MCF-7 cell line (Fig. 5C and D). By contrast, knockdown of MBP1 in the MDA231 and MCF7 cell lines upregulated β-catenin mRNA and protein expression levels (Fig. 5E-H). Lastly, two potential MBP1 binding sites were predicted on the β-catenin promoter. Sites were mutated, and a dual-luciferase assay revealed that MBP1 lost its inhibitory effect on the promoter of β-catenin after the sequence was mutated at site 1 (Fig. 5J). Further investigation into the effect of β-catenin on the relative luciferase activity of MBP1-sh and Lv-MBP1 demonstrated that MBP1 knockdown inhibited β-catenin activity (Fig. 5K) and that MBP1 overexpression promoted β-catenin activity (Fig. 5L). Thus, the results indicated that MBP1 inhibits the transcription of β-catenin in BC cell lines.

Hypoxia promotes tumor development via various mechanisms (21). Therefore, it was investigated whether MBP1 is a hypoxia-responsive factor. Notably, the HIF-1α and β-catenin protein expression levels were increased, while MBP1 protein expression level was decreased in the MDA231 and MCF7 cell lines using western blot analysis following 24 h under hypoxic conditions (1% O₂) (Fig. 6A and B). Similarly, after treatment with the chemoattractant CoCl₂ for 24 h, the HIF-1α and β-catenin protein expression levels were increased, while the protein expression levels of MBP1 was decreased in the MDA231 and MCF7 cell lines (Fig. 6C and D). Furthermore, RT-qPCR revealed no significant difference in the mRNA expression level of MBP1, whereas there were significant differences in the mRNA expression level of β-catenin, under hypoxic (1% O₂) or CoCL₂ conditions in the MDA231 and MCF7 cell lines (Fig. 6E and F), indicating that HIF-1α may not regulate MBP1 expression at the mRNA level, but at the protein level. Thus, Co-IP analysis was performed, using MBP1 and HIF-1α antibodies, to confirm whether MBP1 binds to HIF-1α. MBP1 and HIF-1α could bind to each other (Fig. 6G and H).