Breast cancer (BC) is a common type of tumor. Numerous patients are diagnosed and treated in the early stages of the disease; however, the recurrence rate remains high. Therefore, identifying sensitive and specific tumor markers to prevent and treat BC is essential. c-Myc promoter binding protein 1 (MBP1) is a regulatory molecule located in the cell nucleus. It targets and regulates the expression of various cell proliferation-, apoptosis- and tumor-associated genes. MBP1 expression in BC tissues was detected using immunohistochemistry and further validated in BC and normal human cell lines using RT-qPCR and western blot analysis. Low MBP1 expression, in clinical samples of BC, was associated with a poor prognosis of BC (n=50). MBP1 overexpression effectively inhibited the growth and metastasis of xenograft tumors
Breast cancer (BC) is the most frequent malignant tumor in women worldwide since 2020 and is characterized by occult disease, easy metastasis and recurrence, and a poor prognosis (
With the development of molecular biology, the expression and significance of related tumor markers in cancer are gradually being recognized. The study of tumor markers in BC tissues is increasing (
The present study aimed to investigate the roles and underlying mechanisms of MBP-1 on BC proliferation
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 (
RT-qPCR was performed as previously described (
Western blot analysis was performed as previously described (
IHC was performed as previously described (
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
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
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 (
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 (
To further elucidate the role of MBP1 in BC tumor growth
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 (
β-catenin is a key molecule of the Wnt signaling pathway and a key marker to promote tumor proliferation (
Hypoxia promotes tumor development via various mechanisms (
MBP1 is a tumor suppressor commonly expressed in mammalian cells (
To further investigate the potential mechanisms, the downstream targets of MBP1 were analyzed. β-catenin is a key factor that promotes tumor growth and an important marker of tumor malignant behavior (
In addition, research has found an association between hyponuclear genes and hypoxia regulation, suggesting that HIF-1α is the main direct regulator (
The present study has some limitations. Due to the limited number of clinical samples, IHC or RT-qPCR was not performed to analyze the association between expression of MBP1 and β-catenin or MBP1 and HIF-1α. In addition, RNA sequencing was not performed in BC cell lines with different levels of MBP1 expression; therefore, pathway analysis on downstream targets of MBP1 could not be identified, which will be performed in future studies.
In conclusion, the results indicated that MBP1 could serve as a new biomarker and target to predict the prognosis and clinical treatment of BC.
Not applicable.
The datasets used and/or analyzed during the current study are available in TCGA Genome Data Analysis Center of the Broad Institute (kmplot.com/analysis/). All other datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.
GW conceived and designed the study. YZ participated in the study design and performed bioinformatics analysis. XL, PZ and GP provided their advice during the research process. YZ and XL performed data analysis and wrote the manuscript. YZ, PZ and GP performed cytology experiments. All the authors reviewed and edited the manuscript. All authors have read and approved the final version of the manuscript. All authors confirm the authenticity of all the raw data.
All the clinical samples were collected with written informed consent from the patients, and this protocol was approved by the Ethics Committee of Hubei Cancer Hospital (Wuhan, China). The mouse experiments and handling of the animals were performed according to the Institutional and Animal Care and Use Committee of Hubei Cancer Hospital and the NIH Guide for the Care and Use of Laboratory Animals.
Not applicable.
The authors declare that they have no competing interests.
MBP1 is expressed at low levels in BC compared with adjacent tissues. Expression levels of MBP1 in 50 pairs of BC and N tissues were analyzed using (A) RT-qPCR and (B) immunohistochemistry. The expression levels of MBP1 in breast epithelial cells (MCF10A) and BC cell lines (MDA231, MCF7, MDA468 and BT484) were determined using (C) RT-qPCR and (D) western blot analysis. (E) Expression levels of MBP1 in BC and N tissues from the Protein Atlas database. Scale bar, 50 µm. All the data are presented as the mean ± SD from three independent experiments. **P<0.01. BC, breast cancer; N, normal adjacent tissue; RT-qPCR, reverse transcription-quantitative PCR; MBP1, c-Myc promoter binding protein 1.
Low expression of MBP1 in BC is associated with a poor prognosis. A total of 50 patients with breast cancer were divided into high- and low-MMP1 expression level groups according to (A) RT-qPCR and (B) IHC. Scale bar, 50 µm. (C) The overall survival of the high- and low-expression level groups was analyzed using Kaplan-Meier curves. Using the Kaplan-Meier plotter database the (D) overall survival and (E) disease-free survival, and (F) overall survival times in patients with BC, and in patients with BC receiving endocrine therapy were analyzed, respectively according to the expression level of MBP1. All the data are presented as the mean ± SD from three independent experiments. RT-qPCR, reverse transcription-quantitative PCR; MBP1, c-Myc promoter binding protein 1.
MBP1 inhibits BC growth
MBP1 regulates proliferation of BC cells. mRNA and protein expression level of MBP1 in the MDA231 and MCF7 cell lines transfected with Lv-MBP1 and Lv-NC was determined using (A) RT-qPCR and (B) western blot analysis, respectively. The viability of the (C) MDA231 and (D) MCF7 cell lines was assessed using the CCK-8 assay following transfection with Lv-MBP1 and Lv-NC. (E) The proliferation ability of the MDA231 and MCF7 cell lines was analyzed using a colony formation assay following transfection with Lv-MBP1 and Lv-NC and the results were (F) statistically analyzed. MBP1 mRNA and protein expression level in the MDA231 and MCF7 cell lines transfected with MBP1-sh and NC-sh was determined using (G) RT-qPCR and (H) western blot analysis, respectively. The viability of (I) MDA231 and (J) MCF7 cells was analyzed using the CCK-8 assay following transfection with MBP1-sh and NC-sh. (K) The proliferation ability of the MDA231 and MCF7 cell lines was determined using a colony formation assay following transfection with MBP1-sh and NC-sh and the results were (L) statistically analyzed. All the data are presented as the mean ± SD from three independent experiments. *P<0.05. MBP1, c-Myc promoter binding protein 1; NC, negative control; Lv-MBP1, MBP1 overexpression vector; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin; CCK, Cell Counting Kit.
MBP1 suppresses β-catenin transcription. MBP1 and β-catenin mRNA and protein expression levels were detected using (A) RT-qPCR and (B) western blot analysis, respectively following transfection with Lv-MBP1 and Lv-NC in the MDA231 cell line. MBP1 and β-catenin were detected using (C) RT-qPCR and (D) western blot analysis following transfection with Lv-MBP1 and Lv-NC in the MCF-7 cell line. MBP1 and β-catenin mRNA and protein expression levels were detected using (E) RT-qPCR and (F) western blot analysis, respectively, following transfection with MBP1-sh and NC-sh in the MDA231 cell line. MBP1 and β-catenin mRNA and protein expression levels were detected using (G) RT-qPCR and (H) western blot analysis following transfection with MBP1-sh and NC-sh in the MCF-7 cell line. (I) Potential binding sites between MBP1 and the β-catenin promter, and the 2 MUT sequences. (J) The luciferase activity of the MDA231 cells following cotransfection with β-catenin WT, MUT or empty vector. MDA231 cells were cotransfected with (K) MBP1-sh and NC-sh, and (L) LV-MBP1 and LV-NC. Luciferase activity was detected after transfection for 48 h. All the data are presented as the mean ± SD from three independent experiments. *P<0.05. WT, wild type; MUT, mutant; MBP-1, c-Myc promoter binding protein 1; NC, negative control; Lv-MBP1, MBP1 overexpression vector; sh, short hairpin; reverse transcription-quantitative PCR; n.s., not significant.
MBP1 is regulated by HIF-1α under hypoxic conditions. (A) MDA231 and (B) MCF7 cells were cultured under hypoxic conditions (1% O2) for 24 h, then the protein expression levels of HIF-1α, MBP1 and β-catenin were analyzed using western blot analysis. (C) MDA231 and (D) MCF7 cells were cultured in CoCl2 for 24 h, then the protein expression levels of HIF-1α, MBP1 and β-catenin were detected using western blot analysis. (E) MDA231 and (F) MCF7 cells were cultured under hypoxic conditions (1% O2) or CoCl2 for 24 h, then the mRNA expression levels of MBP1 and β-catenin were detected using reverse transcription-quantitative PCR. Whole cell lysates of the MDA231 cell line were immunoprecipitated with (G) anti-MBP1 or IgG, or (H) anti-HIF-1α or IgG, then subjected to western blot analysis for MBP1 and HIF-1α. All the data are presented as the mean ± SD from three independent experiments. **P<0.01. MBP-1, c-Myc promoter binding protein 1; HIF-1α, hypoxia-inducible factor 1α; n.s., not significant.
Clinical significance of MBP1 in patients with breast cancer.
MBP1 expression | ||||
---|---|---|---|---|
Variable | Number | High | Low | P-value |
Age, years | ||||
<60 | 24 | 12 | 12 | 0.786 |
>60 | 26 | 14 | 12 | |
Tumor size, cm | ||||
<2 | 28 | 18 | 10 | 0.003 |
>2 | 22 | 5 | 17 | |
Histological grade | ||||
High/moderate | 30 | 16 | 14 | 0.006 |
Low | 20 | 3 | 17 | |
TNM stage | ||||
I/II | 28 | 19 | 9 | 0.004 |
III/IV | 22 | 6 | 16 | |
T | ||||
I/II | 29 | 19 | 10 | 0.025 |
III/IV | 21 | 7 | 14 | |
N | ||||
I/II | 17 | 13 | 4 | 0.013 |
III/IV | 33 | 13 | 20 | |
M | ||||
No | 19 | 13 | 6 | <0.001 |
Yes | 31 | 6 | 25 |
T, tumor stage; N, lymph node status; M, metastasis status; MBP-1, c-Myc promoter binding protein 1.