Background
Breast cancer (BC) is the most frequent and highly lethal tumor in women, and triple-negative breast cancer (TNBC) accounts for approximately 10–24% of BCs [
1,
2]. TNBC, the most aggressive type of BC with a high proliferative and metastatic phenotype, is characterized by the absence of expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) [
1,
3]. TNBC has a worse prognosis, higher histological grade and displays high rates of drug resistance, metastasis and prost-surgical reoccurrence [
4‐
7]. To date, there are still no efficient agents for the treatment of TNBC because of a lack of specific therapeutic targets [
8,
9]. Therefore, it is urgent to identify novel specific regulators in TNBC, which might provide new insights and clues for the development of more effective therapies for TNBC.
MicroRNAs (miRNAs) are a class of highly conserved small noncoding single-stranded RNA molecules [
10]. They can regulate a variety of cellular processes, including cell proliferation, mobility, differentiation and metabolism, by base-pairing with the 3′-untranslated regions (3′-UTRs) of target genes [
11]. An increasing number of studies have revealed that miRNA dysfunction contributes to BC development and metastasis [
12,
13]. MiR-33a downregulation has been associated with cancer cell proliferation, mobility and chemotherapy sensitivity in various cancers, including lung cancer [
14,
15], prostate cancer [
16] and BC [
17,
18], which suggested that it functions as a tumor suppressor. MiR-33a was upregulated after treatment with chidamide in TNBC and suppressed glycolysis by targeting LDHA [
19]. It can also target ADAM9 and ROS1 to suppress BC proliferation and metastasis [
18]. However, the detailed mechanisms of miR-33a in TNBC proliferation and mobility remain unclear.
The polycomb group protein enhancer of zeste homolog 2 (EZH2) is a histone-lysine
N-methyltransferase enzyme that regulates DNA methylation and suppresses RNA transcription [
20,
21]. EZH2 is highly expressed in many kinds of cancers including BC [
22], and EZH2 can regulate TNBC tumor growth and metastasis [
23‐
25]. Knockdown of EZH2 suppressed TNBC MDA-MB-231 tumor growth and metastasis in xenograft models [
26,
27]. Tumor-suppressive miRNAs that directly target EZH2 to inhibit TNBC progression include miR-1301 [
28] and miR-340 [
25]. It has also been reported that EZH2 expression in TNBC patients is correlated with aggressiveness, advanced tumor stage and increased mortality [
29,
30]. Therefore. EZH2 inhibition might be a promising potential therapeutic target for the treatment of TNBC. Whether EZH2 expression is associated with miR-33a levels in TNBC has not been determined.
In our study, we analyzed the expression of miR-33a and EZH2 in TNBC tissues and determined the role of miR-33a and EZH2 in TNBC progression. Our resulted uncovered an important role of EZH2 in the growth and mobility of TNBC cells and confirmed EZH2 was a target of miR-33a. Thus, targeting miR-33a/EZH2 signaling may be a potential strategy for TNBC treatment.
Materials and methods
Clinical specimens
TNBC tissues (n = 60) and adjacent nontumor tissues (n = 30), which included 15 TNBC tissues and 15 matched adjacent nontumor tissues from the same patients, were surgically obtained from Guangzhou Panyu Central Hospital. The tissues were stored in liquid nitrogen after collection, and all specimens were confirmed by pathological examination. Prior patient consent and approval from Guangzhou Panyu Central Hospital were obtained for the use of these clinical tissues in the study. This research was authorized by the Ethics Committees of Guangzhou Panyu Central Hospital (ethical number: NN-WK-2016115).
Cells and cell culture
The human triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-453, MDA-MB-468 and BT-549 and human normal breast epithelial cell line MCF10A were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, P.R. China). All the cells were cultured in recommendatory culture media containing 10% FBS and 1% penicillin-streptomycin and maintained in a humidified atmosphere of 5% CO2 at 37 °C.
Cell transfection
For gene knockdown experiments, MDA-MB-231 and BT-594 cells were transfected with EZH2 siRNA (GenePharma) by Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) according to the protocol. For gene overexpression experiments, the cells were transfected with pCMV3-EZH2 plasmid, or miR-33a mimic by Lipofectamine™ 3000 Transfection Reagent (Invitrogen). After a 6-h transfection, medium containing transfection reagents were refreshed and cells were further cultured with fresh medium for 24 h. The knockdown of EZH2 was performed with following siRNA duplex: 5′-GAGGGAAAGUGUAUGAUAATT-3′ and 5′-UUAUCAUACACUUUCCCUCTT-3′.
Quantitative real-time PCR
Total RNA was extracted from TNBC tissues and cell lines using the E.Z.N.A.® Total RNA Kit I (Omega Bio-Tek) according to the manufacturer’s protocol. Reverse transcription was performed with the Transcriptor First Strand cDNA Synthesis Kit (Roche). Then, cDNA was amplified and quantified with the LightCycler 480 Real-Time PCR System (Roche) using 2× SYBR Green I Master Mix (Bimake). For miRNA quantification, total RNA was reverse transcribed with the PrimeSript miRNA cDNA Synthesis Kit (TaKaRa), and the miR-33a cDNA was amplified and quantified with the LightCycler 480 Real-Rime PCR System (Roche) using 2 × SYBR Green I Master Mix (Bimake). The levels of mRNA and miRNA were normalized to ACTB and U6 levels, respectively. The 2−ΔΔCT method was used to determine relative gene expression.
Western blot assay
Total cell protein of TNBC cells was extracted by cell lysis in RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitor cocktails (Bimake). The protein concentrations were detected by a BCA Protein Assay Kit (Invitrogen). Proteins were separated by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) and subjected to immunoblot analyses. The blot was performed with primary antibodies against EZH2 and GAPDH (1:1000 dilution. Cell Signaling Technology). The signals were detected by an HRP-conjugated secondary antibody (1:2000 dilution. Cell Signaling Technology) and the bands were visualized with an enhanced chemiluminescence (ECL, Millipore) system according to the manufacturer’s protocol.
Cell proliferation assay
The effects of miR-33a and EZH2 on the proliferation of MDA-MB-231 and BT-549 cells were measured by the EdU cell proliferation assay (Beyotime Biotechnology) and CCK-8 assay (Beyotime Biotechnology). Briefly, cells were seeded in 96-well plates and cultured overnight. Cells were transfected with miRNA mimic, siRNA or plasmids for 6 h, and the medium was replaced. For the EdU cell proliferation assay, cells were subjected to an EdU cell proliferation assay kit according to the standard protocol at 24 h. The images were acquired with an inverted fluorescence microscope. For the CCK-8 assay, the absorbance at 450 nm was measured using a microplate reader at 0, 24, 48, 72, and 96 h.
Colony formation can be used to evaluate cell proliferation capacity. MDA-MB-231 and BT-549 cells were transfected with miRNA mimic, siRNA or plasmids for 24 h, and then the cells were trypsinized and seeded into 6-well plates at approximately 1000 cells per well. After culture for 10 days, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The visible colonies of cells were observed and counted.
Cell cycle analysis
The effects of miR-33a and EZH2 on the cell cycle in MDA-MB-231 and BT-549 cells were detected using flow cytometry analysis. Cells were transfected with miRNA mimic, siRNA or plasmids for 48 h, trypsinized, fixed with pre-cooled 70% ethanol and treated with 1 mg/mL RNase for 30 min at 37 °C. Then, the intracellular DNA was labeled with propidium iodide (PI) (Beyotime Biotechnology) for 30 min at 4 °C and analyzed by a flow cytometer (BD). The populations of TNBC cells in G1, S and G2/M phases were calculated with ModFit software (Verity Software House Inc., Topsham, ME, USA).
Luciferase reporter assay
The 3′-UTR of EZH2 containing miR-33a binding sites and its mutant were cloned into the pGL3-control luciferase reporter vector. The pGL3-EZH2 or mutant pGL3-EZH2 plasmid was co-transfected with miR-33a or NC mimics into MDA-MB-231 and BT-549 cells. After a 48-h transfection, luciferase activity was evaluated by the Dual-Luciferase Reporter Assay System (Promega) and was normalized to the activity of Renilla luciferase driven by a constitutively expressed promoter in the phRL vector.
Cell migration and invasion assay
The effects of miR-33a and EZH2 on the migration and invasion of MDA-MB-231 and BT-549 cells were measured by Transwell assays (Corning). For the Transwell migration assay, TNBC cells were first transfected with miRNA mimic, siRNA or plasmid. After transfection for 24 h, cells were trypsinized, resuspended in serum-free medium and added into the upper filters. The lower chambers were filled with complete culture medium. After a 24-h incubation, the migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The cells on the inner sides of the upper filters were removed with cotton swabs, and the migrated cells on the bottom sides were imaged. For the invasion assay, the upper filters were precoated with diluted Matrigel (Corning), and the following procedures were the same as those for the Transwell migration assay.
Statistical analysis
Each experiment was repeated three times, and all statistical analyses were performed using GraphPad Prism 8.0 software. Data are presented as the mean ± SEM. The differences between two groups were determined by two-tailed Student’s t test, and differences among more than two groups were analyzed using one-way ANOVA followed by the Tukey test. P < 0.05 indicates a significant difference.
Discussion
Increased evidence supports that noncoding RNAs, including miRNAs, lncRNAs and circRNAs, are important regulators in tumor development, growth, metastasis and angiogenesis [
31,
32]. MiRNAs can be oncogenic miRNAs and tumor-suppressive miRNAs [
33,
34]. It was reported that miR-33a was downregulated in a variety of cancers, and ectopic expression of miR-33a suppressed multiple malignant behaviors [
15‐
18]. miR-33a can suppress epithelial-mesenchymal transition and metastasis in non-small cell lung cancer, and its expression in the blood of lung cancer patients was lower than that in healthy controls; thus, it could be regarded as a tumor suppressor and a novel biomarker for the diagnosis of lung cancer [
15,
35]. It also targets ST8SIA1 to suppress colorectal cancer progression [
36] and targets HIF-1α to inhibit melanoma cell growth and mobility [
37]. Regarding breast cancer, decreased miR-33a in breast cancer tissues was associated with lymph node metastasis, and the expression of miR-33a was dramatically lower in metastatic breast cancer cell lines than in nonmetastatic cancer cell lines and normal breast epithelial cells [
18]. Further study showed that miR-33a overexpression inhibited metastatic breast cancer cell growth and mobility
in vitro and
in vivo by targeting ADAM9 and ROS1 [
18]. Therefore, miR-33a acts as a tumor-suppressive miRNA in most kinds of cancer. However, Wang H reported that upregulated expression of miR-33a was correlated with poor prognosis of GBM patients and enhanced self-renewal of glioma-initiating cells via activation of the PKA and NOTCH pathways by targeting PDE8A and UVRAG [
38]. Thus, miR-33a can act as either oncogenic or tumor-suppressive miRNA, which may depend on the tumor type. Nevertheless, the expression profiles and functions of miR-33a in triple-negative breast cancer growth and metastasis are less clear. In our study, we found that miR-33a was significantly downregulated in TNBC tissues compared with adjacent nontumor tissues, and further studies revealed that ectopic expression of miR-33a inhibited TNBC cell proliferation, colony formation, migration and invasion and induced G1 cell cycle arrest. miR-33a overexpression also downregulated TNBC cell
CDK4 and
MMP9 mRNA levels, which may account for the suppression of proliferation and mobility in TNBC cells.
EZH2, as a methyltransferase and component of PRC2, regulates H3K27 methylation to mediate gene transcriptional silencing [
20]. EZH2 was higher in tumor tissues than in adjacent nontumor tissues and was associated with poor prognosis in tumor patients [
39‐
41]. In breast cancer, its expression was correlated with breast cancer aggressiveness and could serve as an independent predictor of survival and recurrence [
42]. A recent study reported that EZH2 promoted the mobility of TNBC cells by regulating the TIMP-2/MMP-2/9 pathway [
23]. miRNAs were reported to regulate EZH2 expression in TNBC. Wu QJ reported that miR-1301 inhibited TNBC cell proliferation, migration and colony formation as well as xenograft growth by negatively regulating EZH2 expression [
28]. Another study revealed that EZH2 reduction mediated by miR-340 mimic induced decreased expression of DNM1, H3K27me3, β-catenin and p-STAT3, which led to inhibition of miR-21 activity and upregulation of miR-200a/b, which contributed to inhibition of TNBC progression [
25]. Here, our results showed that EZH2 expression was negatively correlated with miR-33a in TNBC tissues and was significantly higher in TNBC cell lines. Moreover, a luciferase reporter assay corroborated that miR-33a directly targeted EZH2 and inhibited EZH2 expression. EZH2 inhibition by EZH2 siRNA exerted similar effects on TNBC cell behaviors, while EZH2 upregulation by transfection with an EZH2-overexpressing plasmid reversed the suppressive effects mediated by miR-33a.
Many factors are responsible for the downregulation of tumor-suppressive miRNAs [
43,
44]. It was reported that miR-33a downregulation in tumors may be associated with upregulation of proto-oncogenic lncRNAs, and this study showed that miR-33a inhibition mediated by lncRNA DANCR promoted osteosarcoma development and cancer stemness characteristics by inducing Axl upregulation [
45]. EZH2 was also reported to induce silencing of tumor-suppressive miRNA I tumor cells, and miR-34a was epigenetically silenced by EZH2, which promoted cholangiocarcinoma cell growth by activating the Notch pathway [
46]. Therefore, whether miR-33a downregulation in TNBC is mediated by EZH2 or oncogenic lncRNA remains to be further investigated.
Given that EZH2 was overexpressed in many types of cancer, and inhibitors targeting EZH2 were also developed. Tazemetostat, an EZH2 inhibitor, has been approved for treating epithelioid sarcoma, and it is the first EZH2 inhibitor approved by FDA [
47]. Other EZH2 inhibitor, GSK343 and GSK236, were also reported to inhibit tumor progression in various cancer, such as glioblastoma [
48], head and neck cancer, [
49] and breast cancer [
50]. Our in vitro study confirmed the regulatory mechanism of miR-33a/EZH2 cascade in TNBC progression, thus, more investigation of the effects of miR-33a and EZH2 on tumor growth and metastasis
in vivo is needed. Moreover, EZH2 inhibitors can be used for in vitro and in vivo studies, which may further provide a rationale for potential therapeutic strategy for the treatment of TNBC patients.
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