Cancer stem cell (CSC) formation and epithelial-mesenchymal transition (EMT) are pivotal events in tumor cell invasion and metastasis. They have been shown to occur in resistance to tamoxifen. Moreover, microRNAs (miRNAs) have been associated with CSCs, EMT as well as tamoxifen resistance. Studying molecular mechanism of CSCs, EMT as well as tamoxifen resistance will help us to further understand the pathogenesis and progression of the disease and offer new targets for effective therapies. In the present study, we showed that miR-375 inhibits CSC traits in breast cancer MCF-7 cells. Bioinformatics analysis and experimental validation identified HOXB3 as a direct target of miR-375. Overexpressing miR-375 degraded HOXB3 mRNA in MCF-7 cells. Moreover, overexpression of HOXB3 induced formation of CSC phenotypes, EMT and tamoxifen-resistance as well as enhanced ability of migration and invasion in MCF-7 cells. Most ER-positive breast cancer-related deaths occur, because of resistance to standard therapies and metastasis, restoring miR-375 or targeting HOXB3 might serve as potential therapeutic approaches for the treatment of tamoxifen-resistant breast cancer.
Approximately 60–70% of breast cancer express estrogen receptor α (ERα) and (or) progesterone receptor and merit the use of hormone therapies, such as the estrogen receptor antagonist tamoxifen (
Emerging evidence suggests a strong link between resistance to therapies and the induction of epithelial-mesenchymal transition (EMT) in cancer (
MicroRNAs (miRs) are small non-coding RNAs that modulate protein expression by binding to complementary or partially complementary the 3-untranslated region (UTR) of target mRNAs and thereby targeting the mRNA for degradation or translational inhibition (
In the present study, we showed that miR-375 inhibits CSC traits in breast cancer MCF-7 cells. Bioinformatics analysis and experimental validation identified HOXB3 as a direct target of miR-375. Overexpressing miR-375 degraded HOXB3 mRNA in MCF-7 cells. Moreover, overexpression of HOXB3 induced formation of CSC phenotypes, EMT and tamoxifen-resistance as well as enhanced ability of migration and invasion in MCF-7 cells. Most ER-positive breast cancer-related deaths occur because of resistance to standard therapies and metastasis, restoring miR-375 or targeting HOXB3 might serve as potential therapeutic approaches for the treatment of tamoxifen-resistant breast cancer.
Human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Briefly, cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2. HOXB3 expressing plasmids/empty vectors (pcDNA3.1) were purchased from Tiangen Biotech, Co., Ltd. (Beijing, China). pre-miR-375/control miR were purchased from Ambion, Inc. (Austin, TX, USA). For transfection experiments, the cells were cultured in serum-free medium without antibiotics at 60% confluence for 24 h, and then transfected with transfection reagent (Lipofectamine 2000; Invitrogen, Carlsbad, CA, USA) according to the manufacturers instructions. After incubation for 6 h, the medium was removed and replaced with normal culture medium for 48 h, unless otherwise specified.
Western blot analysis was performed as previously described (
Cells (103/ml) in serum-free RPMI-1640/1 mM Na-pyruvate were seeded on 0.5% agar precoated 6-well plates. After 1 week, half the medium was changed every third day. Single spheres were picked and counted.
Single cell suspensions of MCF-7 cells transfected as indicated were prepared and plated using ultra low adherent wells of 6-well plate at 5,000 cells/well in sphere formation medium, as described above. After 7 days of treatment, the breast cancer spheres were collected by centrifugation, washed with 1X phosphate-buffered saline (PBS) and fixed with 3.7% parformaldehyde for immunofluorescence staining, as described previously by our laboratory. Coverslips were counterstained with DAPI (Invitrogen-Molecular Probes, Eugene, OR, USA) for visualization of the nuclei. Microscopic analysis was performed with a confocal laser scanning microscope (Leica Microsystems, Bensheim, Germany). Fluorescence intensities were measured in a few viewing areas for 300 cells per coverslip and analyzed using ImageJ 1.37v software (
For immunofluorescence analyses, MCF-7 cells were plated on glass coverslips in 6-well plates and transfected as indicated. At 48 h after transfection, coverslips were stained with HOXB3 (1:500; Abcam) Alexa Fluor 488 goat anti-rabbit IgG antibody was used as secondary antibody (Invitrogen). Similarly to the above, the coverslips were counterstained with DAPI (Invitrogen-Molecular Probes) for visualization of the nuclei. Microscopic analysis was performed with a confocal laser-scanning microscope (Leica Microsystems). Fluorescence intensities were measured in a few viewing areas for 300 cells per coverslip and analyzed using ImageJ 1.37v software (
Migration and invasion assay was performed as described before (
The analysis of potential microRNA target site using the commonly used prediction algorithms, miRanda (
Total RNA from cultured cells, with efficient recovery of small RNAs, was isolated using the mirVana miRNA isolation kit (Ambion). Detection of the mature form of miRNAs was performed using the mirVana qRT-PCR miRNA detection kit and qRT-PCR Primer Sets, according to the manufacturers instructions (Ambion). The U6 small nuclear RNA was used as an internal control.
RT-PCR was performed as described (
The effect of the cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) assay and it was performed as described (
Data are presented as mean ± SEM. Students t-test (two-tailed) was used to compare two groups (P<0.05 was considered significant).
Re-expression of miR-375 can partly reverse EMT in breast cancer (
CD44 is positively associated with stem cell-like characteristics in breast cancer (
Moreover, CD133 has been proposed as a cancer stem cell marker in breast cancer (
Having demonstrated that overexpressing miR-375 inhibited formation of stem cell-like population. Next, we studied the mechanisms of miR-375 regulating CSCs. MicroRNAs (miRs) are a class of small non-coding RNAs (~22 nucleotides) and negatively regulate protein-coding gene expression by targeting mRNA degradation or translation inhibition (
In an attempt to identify the role of miR-375 in regulating HOXB3 expression in breast cancer, we performed immunofluorescence analyses in MCF-7 cells transfected with pre-miR-375 and control miR. The results showed that HOXB3 protein was evidently inhibited in the cells transfected with pre-miR-375 (
In order to study the role of HOXB3 in breast cancer, we transfected MCF-7 cells with HOXB3 expressing plasmids. The results showed that HOXB3 protein could be significantly increased by HOXB3 expressing plasmids in the cells (
To identify whether CD44 protein can be regulated by HOXB3, we performed immunoflurescence to detect CD44 protein in spheres formed by MCF-7 cells transfected with HOXB3 expressing plasmids. We found that CD44 protein level was significantly enhanced by HOXB3 in shperes formed by MCF-7 cells (
To further confirm that CD133 protein can be induced by HOXB3, we performed RT-PCR and western blot analysis to detect CD133 mRNA and protein. The results showed that CD133 mRNA and protein were increased in MCF-7 cells transfected with HOXB3 expressing plasmids (
Re-expression of miR-375 can partly reverse EMT (
Current breast cancer therapeutic strategies based on tumor regression may target and kill differentiated tumor cells which comprise the bulk of the tumor, but the therapeutic strategies spared the rare CSC population. CSCs constitute a mostly small subset of cancer cells that possess the ability to self-renew and generate the diverse differentiated cell populations that constitute the cancer mass (
We found that HOXB3 is a target of miR-375 in MCF-7 cells. The HOX genes encode a family of highly conserved transcription factors that normally regulate temporospatial development of the extremities and organs. Aberrant expression of these genes in different tissues has been associated with tumorigenesis (
Metadherin (MTDH) is a target of miRNA-375, which was upregulated in tamoxifen-resistant cells (
The present study was supported by the grant from the Focus on Research and Development Plan in Shandong Province (2015GSF118132).
cancer stem cells
epithelial-mesenchymal transition
miR-375 inhibits CSC traits in breast cancer MCF-7 cells. (A) Real-time PCR for miR-375 in MCF-7 cells transfected with pre-miR-375 and control miR. U6 was a loading control. n=3. (B) Sphere growth for MCF-7 cells transfected with pre-miR-375 and control miR. The data are reported as the number of spheres formed/1000 seeded cells. Left panel shows microscopic pictures of sphere growth. Right panel shows graphic presentation of average diameter of sphere growth. n=3. (C) Immunofluorescence analyses for CD44 in spheres of MCF-7 cells. Left panel shows microscopic pictures of immunofluorescence staining of one representative experiment (x100 magnification). Right panel shows graphic presentation of mean fluorescence intensities. n=3. (D) Real-time PCR for CD44 in MCF-7 cells transfected with pre-miR-375 and control miR. GAPDH was a loading control. n=3; Western blot analysis for CD44 in MCF-7 cells transfected with pre-miR-375 and control miR. β-actin was a loading control. n=3. (E) Immunofluorescence analyses for CD133 in spheres of MCF-7 cells transfected with pre-miR-375 and control miR. Left panel shows microscopic pictures of immunofluorescence staining of one representative experiment (x100 magnifications). Right panel shows graphic presentation of mean fluorescence intensities. n=3. (F) Real-time PCR for CD133 in MCF-7 cells transfected with pre-miR-375 and control miR. GAPDH was a loading control. n=3; Western blot analysis for CD133 in MCF-7 cells transfected with pre-miR-375 and control miR. β-actin was a loading control. n=3.
miR-375 inhibits HOXB3 in breast cancer MCF-7 cells. (A) Schematic of predicted miR-375 binding sites in the 3UTR of HOXB3 mRNA by miRanda. (B) Immunofluorescence analyses for HOXB3 in MCF-7 cells transfected with pre-miR-375 and control miR (mock). Upper panel shows microscopic pictures of immunofluorescence staining of one representative experiment (x100 magnification). Bottom panel shows graphic presentation of mean fluorescence intensities. n=3. (C) Western blot analysis for HOXB3 in MCF-7 cells transfected with pre-miR-375 and control miR (mock). β-actin was a loading control. n=3. (D) RT-PCR for HOXB3 in MCF-7 cells. MCF-7 cells were transfected with pre-miR-375 and control miR (mock). GAPDH was a loading control. n=3.
HOXB3 promotes CSC phenotypes in MCF-7 cells. (A) Western blot analysis for HOXB3 in MCF-7 cells transfected with HOXB3 expressing plasmids and empty vectors (mock). β-actin was a loading control. n=3. (B) Sphere growth for MCF-7 cells transfected with HOXB3 expressing plasmids and empty vectors (mock). The data are reported as the number of spheres formed/1000 seeded cells. Left panel shows microscopic pictures of sphere growth. Right panel shows graphic presentation of average diameter of sphere growth. n=3. (C) Immunofluorescence analyses for CD44 in spheres of MCF-7 cells transfected with HOXB3 expressing plasmids and empty vectors (mock). Left panel shows microscopic pictures of immunofluorescence staining of one representative experiment (x100 magnification). Right panel shows graphic presentation of mean fluorescence intensities. n=3. (D) Real-time PCR for CD44 in MCF-7 cells transfected with HOXB3 expressing plasmids and empty vectors (mock). GAPDH was a loading control. n=3; Western blot analysis for CD44 in MCF-7 cells transfected with HOXB3 expressing plasmids and empty vectors (mock). β-actin was a loading control. n=3. (E) Immunofluorescence analyses for CD133 in spheres of MCF-7 cells transfected with HOXB3 expressing plasmids and empty vectors (mock). Left panel shows microscopic pictures of immunofluorescence staining of one representative experiment (x100 magnifications). Right panel shows graphic presentation of mean fluorescence intensities. n=3. (F) Real-time PCR for CD133 in MCF-7 cells transfected with pre-miR-375 and control miR (mock). GAPDH was a loading control. n=3; Western blot analysis for CD133 in MCF-7 cells transfected with pre-miR-375 and control miR (mock). β-actin was a loading control. n=3.
HOXB3 promotes EMT and tamoxifen resistance in MCF-7 cells. (A) Phase-contrast images of MCF-7 cells transfected with HOXB3 expressing plasmids and empty vectors (mock). n=3. (B) Migration and invasion assays for MCF-7 cells transfected with HOXB3 expressing plasmids and empty vectors (mock). n=3 (C) MTT for cell viability in MCF-7 cells. MCF-7 cells transfected with HOXB3 expressing plasmids and empty vectors (mock) were untreated or treated with tamoxifen. (D) Western blot analysis for MTDH in MCF-7 cells transfected with pre-miR-375 and control miR (mock). β-actin was a loading control. n=3. (E) Western blot analysis for TWIST in MCF-7 cells transfected with pre-miR-375 and control miR (mock). β-actin was a loading control. n=3.
The mechanism of miR-375 as tumor suppressive gene in breast cancer.