1 Introduction
Breast cancer is the most commonly diagnosed cancer in women (23 % of all cancers), with an estimated diagnosis of 1.38 million new cases each year worldwide [
1‐
4]. At the molecular level, breast cancers can be classified in four main subtypes, depending on the expression of hormone and growth factor receptors. The luminal subtypes A and B express both the estrogen receptor (ER) and the progesteron receptor (PR), the HER2-enriched subtype shows amplification and over-expression of the human epidermal growth factor receptor 2 (HER2), and the triple-negative subtype does not express any of these receptors. This classification correlates well with the prognosis, ranging from good for luminal A tumors to poor for the triple-negative tumors [
5,
6]. The latter category, representing ~10 to 15 % of all breast cancers, is generally considered aggressive [
7,
8].
Triple-negative breast cancers are often linked to inactivation of the
BRCA1 tumor suppressor gene. In familial breast cancer, 85 % of the
BRCA1 mutated tumors are triple-negative [
9,
10]. In sporadic triple-negative breast cancers,
BRCA1 is frequently inactivated at the transcriptional level, and it has been shown that
BRCA1 inactivation may be due to methylation of its promoter brought about by ID4 [
11,
12]. More recently it has been found that
BRCA1 may also be regulated at the post-transcriptional level by microRNAs [
13].
MicroRNAs (miRNAs) are small non-coding 19–25 nucleotide-long RNAs that can post-transcriptionally regulate gene expression by binding to the 3’untranslated regions (3′-UTR) of target messenger RNAs (mRNAs), thereby leading to mRNA degradation or translational repression [
14‐
16]. miRNAs have been shown to be involved in diverse biological processes [
17‐
19]. In human breast cancer it has been shown that they can act either as tumor suppressors (i.e., miR-206, miR-17-5p, miR-125a, miR-125b, miR-200, let-7, miR-34 and miR-31) or as oncogenes (i.e., miR-21, miR-155, miR-10b, miR-373 and miR-520c) [
20]. Previously, it has been shown that
BRCA1 gene expression is relatively low in ER-negative and high-grade breast cancers [
21] and that
BRCA1 gene expression is significantly down-regulated in triple-negative breast cancers [
22]. The number of miRNAs that may regulate the expression of
BRCA1 or that may serve as transcriptional targets of
BRCA1 is rapidly increasing. Considering the tumor-suppressive role of
BRCA1, any perturbation in this regulatory function is likely to have an effect on
BRCA1-mediated tumor development [
23]. Moskwa et al. reported that miR-182-mediated down-regulation of
BRCA1 can impede DNA repair and, as such, affect breast cancer therapy [
24]. It has also been shown that miR-146a and miR-146b-5p can down-regulate the expression of the
BRCA1 gene in triple-negative sporadic breast cancers [
18]. In contrast, a recent study reported that miR-146a expression levels increased simultaneously with
BRCA1 expression levels. In addition, it was suggested that post-transcriptional regulation of epidermal growth factor receptor (
EGFR) expression by
BRCA1 may be mediated by miR-146a [
25].
Here, we investigated miRNA expression profiles in nine human breast cancer-derived cell lines and one benign breast epithelium-derived cell line. By doing so, we identified a set of miRNAs putatively involved in BRCA1 gene expression regulation and breast cancer development.
2 Materials and methods
2.1 Cell culture
Nine selected human breast cancer-derived cell lines and one benign breast epithelium-derived cell line (Table
1) were cultured in a humified incubator at 37 °C containing 5 % CO2. Cell line MCF10a was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and was cultured in DMEM/F12 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10 % horse serum, 20 ng/ml EGF, 100 ng/ml cholera toxin, 500 ng/ml hydrocortisone, 2 mM L-glutamine and 20 ng/ml gentamicin.
Table 1
Human breast cancer cell lines and their characteristics
MCF10A | Non tumoral | Benign | wild-type | – | – | – | + (90 %) | + (80 %) | + (100 %) | – |
MCF7 | Tumoral | Luminal | wild-type | 90 % (1+) | 40 % (1/2+) | – | – | – | – | – |
T47D | Tumoral | Luminal | wild-type | 50 % (1/2+) | 80 % (1/2/+) | – | – | – | +(40 %) | – |
MDA-MB-231 | Tumoral | TN | wild-type | – | – | – | – | – | + (100 %) | – |
HCC1937 | Tumoral | TN | 5382insC | – | – | – | + (25 %) | + (20 %) | + (100 %) | – |
MDA-MB-436 | Tumoral | TN | 5396 + 1G > A | – | – | – | – | – | + (90 %) | – |
SUM149PT | Tumoral | TN | 2288delT | – | – | – | + (25 %) | + (<1 %) | + (100 %) | – |
SUM1315MO2 | Tumoral | TN | 185delAG | – | – | – | – | – | + (90 %) | – |
SUM1315-LXSN | Tumoral | TN | 185delAG | – | – | – | – | – | + (60 %) | – |
SUM1315-BRCA1 | Tumoral | TN | 185delAG + WT | – | – | – | – | – | + (80 %) | – |
Cell lines MCF7, T47D, MDA-MB-231, HCC1937 and MDA-MB-436 were also obtained from the ATCC and were cultured in RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS), 2 mM L-glutamine and 20 ng/ml gentamicin. Cell lines SUM149PT and SUM1315MO2 were obtained from Asterand (Royston, Hertfordshire, UK) and were grown in Ham’s F12 medium according to the supplier’s instructions. Cell lines SUM1315-LXSN and SUM1315-BRCA1 were derived from the
BRCA1 mutated (185delAG) SUM1315MO2 cell line, stably transfected with empty LXSN or LXSN-BRCA1 plasmids, respectively. The
BRCA1 mutation status of the other cell lines has previously been reported by
Elstrodt et al. [
26].
2.2 Immunocytochemistry
Cells were fixed in Preservcyt solution (Thinprep) and cytoblocks were prepared using a Shandon Cytoblock kit (Thermo Scientific). The estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin5/6, cytokeratin14, epidermal growth factor receptor (EGFR) and tyrosine kinase receptor cKIT protein status was determined by immunocytochemistry on 3 μm-thick sections. Immunostainings were performed in a BenchmarkXT fully automatized stainer (Ventana Medical Systems) and scored semi-quantitatively by an expert pathologist under a light microscope.
2.3 MiRNA target prediction algorithms
For all algorithms we used the default parameters and we selected miRNAs that putatively target BRCA1 by at least three algorithms.
Total cell RNA was extracted using QIAzol reagent (Qiagen miRNeasy mini kit) according to the manufacturer’s instructions. The RNA quality and yields were assessed using an Agilent Bioanalyzer 2100 (Kit Agilent RNA 6000 nano) in conjunction with a spectrophotometer. One microgram of total RNA was reverse-transcribed using a miscript II RT kit (Qiagen, France). 15 nanograms of cDNA was used in triplicate for quantitative real-time PCR using a miscript SYBER Green PCR kit (Qiagen, France) on an ABI7900HT system and an Applied Biosystems ViiA™ 7 Real-Time PCR system. Selected miRNA primers were obtained from miScript Primer Assay (Qiagen, France) and are listed in supplementary Table
S1. U6 (Hs-RNU6-2–1 miscript Primer Assay) and 18S rRNA were used as internal controls. All samples were normalized to the internal controls and fold changes were calculated through relative quantification (RQ = 2
-∆∆CT).
2.5 PCR array-based profiling
The Human Breast Cancer miScript miRNA PCR Array (MTHS-109ZE-4) was used according to the manufacturer’s protocol (Qiagen, France) for the profiling of 84 miRNAs known or predicted to alter in expression during breast cancer initiation and/or progression. The protocol enables real-time PCR profiling of mature miRNA on an ABI7900HT system in combination with a miScript SYBR Green PCR kit, which contains a miScript Universal Primer (reverse primer), and a QuantiTect SYBR Green PCR Master Mix. 250 nanogram of total RNA was reverse-transcribed using a miscript II RT kit (Qiagen).
2.6 Transfection assays
Selected miRNA mimics and miRNA inhibitors (‘antagomirs’) were obtained from miScript miRNA Mimic and miScript miRNA Inhibitor, respectively (Qiagen, France) and are listed in supplementary Table
S2. The siRNA used for inhibiting
BRCA1 gene expression (siBRCA1) was synthesized by Thermo Scientific, Dharmacon (M-003,461–02–0005, siGENOME SMART pool, Human BRCA1 (672), 5 nmol). Cells were seeded at a density of 300,000 cells per well in 6-well plates. Twenty-four hours later, miRNAs and siRNAs were transfected using Lipofectamine 2000 reagent (Invitrogen, CA, USA) following the manufacturer’s instructions. As a mock control, cells were transfected with transfection reagent alone (Tmock). Three days after transfection, the cells were washed with PBS and recovered for further analysis.
2.7 Cell proliferation assay
MDA-MB-231 cells and MCF7 cells were collected, seeded at a density of 3000 cells per well into a 96-well plate and cultured in a humified incubator at 37 °C containing 5 % CO2 for 24 h. Subsequently, the cells were transfected with Tmock (transfection reagent alone), siBRCA1, miR-146a, anti-miR-146, miR-153, miR-10b and miR-26a using Lipofectamine 2000 reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. After 48 h in vitro cell proliferation was evaluated using CCK-8 (Cell Counting Kit-8, Sigma-Aldrich) according to the manufacturer’s instructions. The absorbance was determined at 450 nm using a microplate reader. All experiments were performed in triplicate.
2.8 In silico analysis of miRNAs using TCGA
Both clinical and miRNA sequencing data of invasive breast cancers were downloaded from The Cancer Genome Atlas (TCGA) database. A total of 519 patients with information on ER, PR and HER2 status were selected to compare the expression profiles of four selected miRNAs (miR-10b, miR-26a, miR-146a and miR-153) in the respective tumors. 88 cases were found to have a negative ER, PR and HER2 (i.e., triple-negative) phenotype, whereas 431 cases were positive for at least one of these receptors.
2.9 Statistical analysis
Student’s t-test was used to assess statistical differences in mean expression between groups. A p-value ≤ 0.05 was considered statistically significant.
4 Discussion
Triple-negative breast cancers, representing ~10 to 15 % of all human breast cancers, have gained growing interest in recent years [
32]. In addition, the role of microRNAs (miRNAs) in the epigenetic regulation of many cellular processes has increasingly become recognized as an important way to fine-tune gene expression [
33]. Here, expression profiling, transfection and
in silico assays were performed to identify potential triple-negative breast cancer miRNA biomarkers, i.e., miR-10b, miR-26a, miR-146a and miR-153.
We found that miR-10b was higher expressed in triple-negative breast cancer-derived cell lines compared to luminal breast cancer-derived cell lines. TCGA data, however, indicated that the average expression of miR-10b was significantly lower in triple-negative tumors than in non triple-negative tumors. O’Day and Lal reported that miR-10b was over-expressed only in metastatic cancer cells, and was found to promote tumor cell migration, invasion and metastasis
in vivo [
20]. Whereas miR-10b has been found to be highly expressed in several human cancers [
34], Biagioni et al. found that miRNA-10b was down-regulated in tumor tissues compared to their normal matched counterparts due to hypermethylation of CpG islands upstream from the miR-10b/10b* locus [
35]. We found that miR-10b can down-regulate the expression of
BRCA1 in MDA-MB-231 and MCF7 cells. High miR-10b expression may thus be a way to silence
BRCA1 expression in
BRCA1 wild-type cells. Recently, it was reported that miR-10b may also play a critical role in TGF-induced epithelial-mesenchymal transition (EMT) in breast cancer and, as such, may be considered as a possible therapeutic target [
36]. Moreover, miR-10b was shown to be able to promote the invasion and metastasis of tumor cells through post-transcriptional regulation of
HOXD10 [
37]. Overall, these results indicate that miR-10b may act as an oncogenic miRNA in breast cancer cells. Nevertheless, Biagioni et al. reported that exogenous miR-10b expression reduced both the
in vitro and
in vivo proliferative capacities of MCF7 and MDA-MB-468 cells [
35]. Our results are compatible with those of Biagioni et al., as we found that miR-10b significantly inhibited the proliferation of MCF7 cells (
p = 0.006). Thus, restoration of miR-10b expression may hold a therapeutic promise for breast cancer treatment.
Another potential biomarker of triple-negative breast cancer that we identified is miR-26a, and we found that miR-26a can down-regulate the expression of
BRCA1 in MDA-MB-231 and MCF7 cells. These results suggest an oncogenic function of this miRNA in breast cancer. In contrast, Gao et al. reported that miR-26a could inhibit the proliferation and migration of breast cancer cells through repression of
MCL-1, and that miR-26a could increase the sensitivity of breast cancer cells to paclitaxel [
38]. Others found that miR-26a can promote ovarian cancer cell proliferation by targeting
ERα [
39]. We found that miR-26a significantly stimulated the proliferation of MDA-MB-231 cells and inhibited the proliferation of MCF7 cells. We also found that the average expression of miR-26a was not significantly different between triple-negative and non triple-negative breast tumors in the TCGA database. The role of miR-26a thus appears to be complex and may dependent on the tissue and/or tumor context.
Our cell line and TCGA data analyses show that miR-146a is significantly over-expressed in triple-negative breast cancers. Similar results were reported by others [
18,
25]. We also found that miR-146a does not affect the expression of
BRCA1 in MDA-MB-231 and MCF7 cells. In contrast, Gracia et al. reported that miR-146a can down-regulate the expression of
BRCA1 in triple-negative sporadic breast cancers [
18]. This discrepancy may be due to the different cell lines used for the exogenous expression assays, i.e., Gracia et al. used HeLa cells and three mammary cell lines (MDA-MB-468, MDA-MB-157 and MDA-MB-436). Another study reported that miR-146a can inhibit the invasion and migration of the highly metastatic human breast cancer cell line MDA-MB-231 [
40]. These data are in line with our results showing that miR-146a can inhibit the proliferation of MDA-MB-231 cells. In a recent study it was hypothesized that BRCA1 may down-regulate the expression of the
EGFR gene by increasing the miR-146a level in breast cancer-derived cells (HCC1937) [
25]. Our results are compatible with this hypothesis, as we found an increase in miR-146a expression in
BRCA1 wild-type SUM1315-BRCA1 cells compared to
BRCA1 mutated SUM1315-LXSN cells. We also found that exogenous expression of miR-146a decreased the proliferation of MDA-MB-231 cells that highly express
EGFR, but not in MCF7 cells that do not express
EGFR. qRT-PCR assessment of
EGFR expression in exogenous miR-146a expressing MDA-MB-231 and MCF7 cells revealed that the
EGFR gene was not significantly regulated by this miRNA. Taken together, these results suggest a complex feedback regulation between miR-146a and
BRCA1, which again most likely depends on the cellular context.
Using bioinformatic tools, we identified a binding site for miR-153 in the 3′-UTR of
BRCA1. Subsequently, we found that miR-153 can induce
BRCA1 down-regulation in MCF7 cells and up-regulation in MDA-MB-231 cells. These contradictory results reflect those reported in the literature. On one hand, Anaya-Ruiz et al. reported that silencing of miR-153 significantly inhibited the growth, reduced the proliferation and induced apoptosis in the triple-negative sporadic breast cancer-derived cell line MDA-MB-231. These results indicate that miR-153 may function as an oncogenic miRNA, whose deregulation could be involved in the initiation and/or development of human breast cancer [
41]. In another report, Wu et al. suggested that miR-153 may play an important role in promoting the proliferation of human prostate cancer cells and, as such, may represent a novel mechanism of miRNA-mediated
PTEN silencing in prostate cancer cells [
42]. These data are compatible with our finding that
BRCA1 up-regulation by miR-153 had no effect on the proliferation of MDA-MB-231 cells. On the other hand, Zhao et al. found that transient transfection of miR-153 into glioblastoma multiforme stem cells (GBM-SCs) can inhibit their stemness properties, repress their growth potential and induce apoptosis [
43]. These latter data are consistent with our results in MCF7 cells: although
BRCA1 was down-regulated by miR-153, it inhibit proliferation. This difference in response observed between MDA-MB-231 and MCF7 cells may be explained by differences in the endogenous levels of miR-153, i.e., higher in luminal MCF7 cells and lower in triple-negative MDA-MB-231 cells. TCGA data analysis also indicated that the average expression of miR-153 is significantly lower in triple-negative breast cancers. We conclude that also the function of miR-153 is complex, and identifying targets of miR-153 would be an interesting means to better understand its role in cell proliferation and carcinogenesis-related processes.
In conclusion, we identified miR-10b, miR-26a, miR-146a and miR-153 as potential triple-negative breast cancer biomarkers. These miRNAs may be instrumental for the future design of novel targeted treatment options of these breast cancers. This ‘from bench-top to bed-side’ translation remains, however, challenging [
44] and additional research is required to better understand the function(s) of these miRNAs.