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Introduction

Breast cancer is one of the most frequently diagnosed malignancies among women (1). During the past few decades, the incidence of breast cancer has continued to increase, and a total of 2,088,849 newly diagnosed cases of breast cancer were reported in 2018 worldwide (2,3). The majority of patients with breast cancer are curable at the non-metastatic stage; however, current therapeutics exhibit minimal efficacy against advanced-stage metastatic disease. Based on transcriptional profiling studies, breast cancer has been established as a heterogeneous malignancy comprising different subtypes, including ductal carcinoma, lobular carcinoma, fibroadenoma and ductal carcinoma in situ (4,5). Based on the molecular characteristics, breast cancer may be divided into hormone receptor-positive breast cancer, in which breast cancer cells express estrogen receptor (ER) or progesterone receptor (PR), or triple-negative breast cancer (TNBC), in which breast cancer cells are negative for ER, PR and human epidermal growth factor receptor 2 (HER2) expression. Although TNBC only accounts for 15-20% of all breast cancer cases, its aggressive nature and the current lack of effective therapies make it one of the most lethal malignancies. Therefore, further understanding the underlying mechanisms involved in TNBC may help with the development of effective therapeutic strategies.

MicroRNAs (miRNAs/miRs) are small non-coding RNAs, 19-26 nucleotides in length, that have no protein-coding ability. miRNAs bind to target mRNAs to suppress their translation (6). Accumulating evidence indicates that miRNAs serve an important role in tumorigenesis, as they are commonly found to be aberrantly expressed in cancer tissues. The dysregulated expression of miRNAs results in uncontrolled cell proliferation, growth and migration in numerous different types of cancer through regulating the expression of downstream tumor suppressor genes or oncogenes. miR-301a-3p has been recognized as a regulator of the T-helper 17 cell immune response in autoimmune demyelinating diseases (7). In addition, miR-301a-3p was previously demonstrated to play an oncogenic role in various types of cancer, including breast cancer (8-13). However, to the best of our knowledge, the downstream target genes of miR-301a-3p in TNBC have not been identified to date.

The present study was undertaken to determine the significance of miR-301a-3p in TNBC. The expression levels of miR-301a-3p were analyzed in normal breast MCF-10A cells and in MDA-MB-231 TNBC cells. The effects of miR-301a-3p on the viability and migratory ability of MDA-MB-231 cells were then examined, and the role of mesenchyme homeobox 2 (MEOX2) in this process was also investigated. The aim was to determine whether miR-301a-3p acts as an oncogenic miRNA in TNBC and whether its effects are mediated by regulating MEOX2 expression.

Materials and methods

Patient studies

Human breast cancer tissues were collected from patients who were diagnosed with TNBC and non-TNBC by three independent pathologists. The patients were enrolled between March 2016 and May 2020 at the Beijing Obstetrics and Gynecology Hospital (Beijing, China). The protocol of the present study was approved by the Clinical Research Ethics Committee of Beijing Obstetrics and Gynecology Hospital, and written, informed consent was obtained from each patient for the use of their tissue prior to participation.

The Cancer Genome Atlas (TCGA) data analysis

Normalized transcriptome expression datasets for breast cancer from the TCGA were analyzed using the ENCORI Pan-Cancer Analysis Platform (http://starbase.sysu.edu.cn/index.php). Briefly, a total of 1,085 breast cancer tissues and 104 normal tissues were available for miR-310a-3p expression level analysis.

Cell lines and culture

MCF-10A normal human breast epithelial cells and MDA-MB-231 TNBC cells were purchased from the American Type Culture Collection. MDA-MB-231 and MCF-10A cells were cultured in RPMI-1640 and DMEM/F12 medium (HyClone; Cytiva), respectively, supplemented with FBS (Gibco; Thermo Fisher Scientific, Inc.) and penicillin-streptomycin solution (Corning, Inc.). The cells were maintained at 37˚C in an atmosphere containing 5% CO2.

Cell transfection

MDA-MB-231 cells were plated into 6-well plates and, upon reaching 70-80% confluence, the cells were transfected with 20 µM miR-301a-3p mimic, mimic negative control (NC), miR-301a-3p inhibitor and inhibitor NC (all purchased from Guangzhou RiboBio Co. Ltd.) using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following 48 h of transfection, cells were collected for use in further experiments. The sequences of the oligonucleotides were as follows: miR-301a-3p mimic, 5'-GCUCUGACUUUAUUGCACUACU-3'; mimic NC, 5'-UCACAACCUCCUAGAAAGAGUAGA-3'; miR-301a-3p inhibitor, 5'-AGUAGUGCAAUAAAGUCAGAGC-3'; and inhibitor NC, 5'-UCUACUCUUUCUAGGAGGUUGUGA-3'. For MEOX2 knockdown, cells were transfected with 40 nM small interfering (si)RNA targeted to MEXO2 (5'-AAGGUAGGACAUGUGGUCAGAUCUU-3') and NC (5'-AAUUAGCGGAGGCUAUAAGAUGUUC-3'), all purchased from Guangzhou RiboBio Co., Ltd. using RNAiMAX reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The full-length open reading frame of the human MEOX2 gene (NM_005924.5) was synthesized and cloned into pcDNA3.1 plasmid. pcDNA3.1 MEOX2 or empty vector (3 µg for 6-well plates) was transfected into MDA-MB-231 cells using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following 48 h of transfection, cells were collected for use in further experiments.

Reverse transcription-quantitative PCR (RT-qPCR) analysis

MDA-MB-231 cells were washed with PBS and lysed in 0.5 ml RNAiso Plus (Takara Biotechnology Co., Ltd.). Following incubation for 10 min on ice, 0.1 ml trichloromethane was added to the lysates and total RNA was isolated. Total RNA was reverse-transcribed into cDNA, using the PrimeScript RT-PCR kit according to the manufacturer's instructions (Takara Biotechnology Co., Ltd.). qPCR was subsequently performed using a SYBR Green PCR kit (Takara Biotechnology Co., Ltd.). The thermocycling conditions were as follows: 94°C for 3 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec, with a final extension step at 72°C for 10 min. U6 and β-actin served as internal controls for miR-301a-3p and MEOX2 expression levels, respectively. The following primer sequences were used for the qPCR: MEOX2 forward, 5'-TCAGAAGTCAACAGCAAACCCAG-3' and reverse, 5'-TTCACCAGTTCCTTTTCCCGAG-3'; β-actin forward, 5'-GACCTGACTGACTACCTCATGAAGAT-3' and reverse, 5'-GTCACACTTCATGATGGAGTTGAAGG-3'; miR-301a-3p, forward 5'-CGTGCGAAGCTCAGGAGGG-3' and reverse, 5'-TGGCTGTCGTGGACTGCG-3'; and U6 forward 5'-CTCGCTTCGGCAGCACA-3' and reverse, 5'-AACGCTTCACGAATTTGCGT-3'. RNA expression levels were normalized to U6 and β-actin. The 2-ΔΔCq method was performed to determine the relative expression (14).

Western blotting

Total protein was extracted from MDA-MB-231 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a BCA assay kit, and protein lysates were then suspended in loading buffer and boiled at 98˚C for 10 min. Subsequently, 30 µg protein/lane was separated via 10-12% SDS-PAGE and separated proteins were transferred onto PVDF membranes, which were then blocked in 5% skimmed milk at 4°C overnight. After washing with TBS, the membranes were incubated with the following primary antibodies at 4-8˚C for ≥12 h: Anti-MEOX2 (1:1,000; cat. no. P50222; RayBiotech, Inc.) and β-actin (1:1,000; cat. no. GTX11003, ProteinTech Group, Inc.). Following primary antibody incubation, the membranes were washed with PBS-Tween-20 (0.1%) thrice and incubated with the corresponding secondary antibodies [HRP-conjugated goat anti-mouse IgG (H + L), 1:2,000; cat. no. AS003; ABclonal Biotech Co., Ltd.] at room temperature for 2 h. Protein bands were visualized using an ECL-Plus kit (Thermo Fisher Scientific, Inc.).

Cell viability assay

MDA-MB-231 cell viability was determined using an MTT assay. Briefly, 5x103 cells/well were seeded into 96-well plates and cultured for 72 h. Subsequently, MTT (5 mg/ml) was added into each well and incubated for a further 3 h. The culture medium was subsequently discarded and 100 µl DMSO was added to each well to dissolve the purple formazan crystals. The optical density value was measured at a wavelength of 595 nm to analyze cell viability.

Flow cytometric analysis of apoptosis

Cell apoptosis was analyzed using a propidium iodide (PI)/Annexin V-FITC kit (Beyotime Institute of Biotechnology). Briefly, MDA-MB-231 cells were digested with EDTA-free trypsin and centrifuged at 157 x g for 5 min at room temperature, then the cell pellet was washed twice with PBS. Subsequently, 0.5-1x105 cells were dissolved in binding buffer and incubated with 5 µl Annexin V-FITC and 10 µl PI. Apoptotic cells were analyzed using a BD Accuri™ C6 Plus flow cytometer (BD Biosciences) using BD Accuri™ C6 software (BD Biosciences), after which the flow cytometric data were analyzed using a Guava easyCyte flow cytometer (EMD Millipore).

Cell invasion and migration assays

Transwell plates with or without Matrigel precoating were used to determine cell invasion and migration, respectively. The upper surface of the chamber was precoated with 15 µl of Matrigel (BD Biosciences) at room temperature for the cell invasion assay. A total of 1.5x105 MDA-MB-231 cells were plated into 8.0-µm pore Transwell chambers (BD Biosciences) and cultured at 37˚C with 5% CO2. The upper chamber was incubated with FBS free RPMI-1640 and DMEM/F12 medium, repsctively, whereas a total of 500 µl 10% FBS medium was added in the lower chamber. Following 24 h of incubation, cells in the upper chamber were removed by cotton-tipped swabs, while cells in the lower chamber were fixed with 100% methanol for 30 min at room temperature and stained with 0.2% crystal violet solution at room temperature. The chambers were then washed with PBS and dried at room temperature. Migratory or invasive cells were visualized under an inverted microscope (magnification, x200; Olympus Corporation).

Wound healing assay

Wound healing assay was performed to analyze the cell migratory ability. Briefly, 2x105 MDA-MB-231 cells were seeded into 6-cm culture plates and cultured in RPMI-1640 medium supplemented with 10% FBS for 48 h. Upon cells reaching 90% confluence, a 200-µl pipette tip was used to create a single linear scratch in the middle of the cell monolayer. The medium was removed, and the cells were incubated with RPMI-1640 medium containing 1% FBS. The wound was visualized using an inverted microscope (magnification, x200; Olympus Corporation) and images were captured at 0 and 24 h.

Dual luciferase reporter assay

Wild-type (WT) or mutant (MUT) 3'-untranslated region (UTR) sequences of MEOX2 were cloned into psi-CHECK vectors (Promega Corporation). Cells were subsequently transfected with the aforementioned vectors and co-transfected with miR-301a-3p mimic (5'-GCUCUGACUUUAUUGCACUACU-3') or negative control (5'-UCACAACCUCCUAGAAAGAGUAGA-3') (Beijing SyngenTech Co., Ltd.) using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After transfection for 48 h, relative luciferase activity was measured using a Dual-Luciferase Reporter assay system (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software (version 8.0; GraphPad Software, Inc.) and data are presented as the mean ± SEM. Statistical differences between two groups were analyzed using unpaired Student's t-test, while statistical differences between multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-301a-3p increases the viability of MDA-MB-231 cells

The expression levels of miR-301a-3p were upregulated in breast cancer tissues in datasets from the TCGA database (Fig. 1A). In addition, miR-301a-3p expression levels were found to be upregulated in TNBC tissues compared with normal breast tissue and luminal type breast cancer tissue according to the TCGA database (Fig. 1B). No statistically significant differences were observed in miR-301a-3p expression levels between TNBC and HER+ breast cancer samples (Fig. 1B). The expression levels of miR-301a-3p in samples from patients with and without TNBC were subsequently analyzed. RT-qPCR analysis revealed that the expression levels of miR-301a-3p were upregulated in TNBC tissues compared with adjacent non-TNBC tissues (Fig. 1C). Subsequently, the expression levels of miR-301a-3p in normal MCF-10A breast epithelial cells and MDA-MB-231 TNBC cells were determined. miR-301a-3p expression levels were found to be upregulated in MDA-MB-231 cells compared with those in MCF-10A cells (Fig. 1D). Thus, MDA-MB-231 cells were selected to further investigate the role of miR-301a-3p in TNBC. The cells were divided into five experimental groups: i) Untreated; ii) miR-301a-3p mimic; iii) mimic NC; iv) miR-301a-3p inhibitor; and v) inhibitor NC. The transfection of cells with the miR-301a-3p mimic or inhibitor led to significantly increased and decreased miR-301a-3p expression levels, respectively, compared with the respective NCs (Fig. 2A). No statistically significant differences in cell viability were observed among the untreated, NC mimic-transfected and NC inhibitor-transfected MDA-MB-231 cells (Fig. 2B). However, miR-301a-3p overexpression increased MDA-MB-231 cell viability, whereas miR-301a-3p knockdown exerted the opposite effect on MDA-MB-231 cells (Fig. 2B). These findings suggested that miR-301a-3p may serve as an oncogenic miRNA in TNBC.

Figure 1 miR-301a-3p expression is upregulated in breast cancer tissues and cells. (A) miR-301a-3p expression was analyzed in breast cancer tissues from TCGA database. P<0.01. (B) miR-301a-3p expression in breast cancer subclasses was analyzed from TCGA database. Normal vs. TNBC, luminal and HER2-positive, all ***P<0.001. Luminal vs. TNBC, **P<0.01. HER2-positive vs. TNBC, not significant. Luminal vs. HER2-positive, *P<0.05. (C) RT-qPCR analysis of miR-301a-3p in non-TBNC and TBNC tissues. ***P<0.001. (D) RT-qPCR analysis of miR-301a-3p expression in MCF-10A and MDA-MB-231 cells. ***P<0.001. TCGA, The Cancer Genome Atlas; miR, microRNA; BC, breast cancer; TNBC, triple-negative breast cancer; HER2, human epidermal growth factor receptor 2; RT-qPCR, reverse transcription-quantitative PCR.

Figure 2 miR-301a-3p regulates the proliferation and apoptosis of MDA-MB-231 cells. (A) MDA-MB-231 cells, untreated (blank) or transfected with mimics NC, mimics miR-301a-3p, inhibitor NC and inhibitor miR-301a-3p were subjected to reverse transcription-quantitative PCR analysis of miR-301a-3p expression. *P<0.05 vs. NC. (B) MDA-MB-231 cells, untreated (blank) or transfected with mimics NC, mimics miR-301a-3p, inhibitor NC and inhibitor miR-301a-3p were subjected to MTT analysis of cell proliferation. *P<0.05 vs. mimics NC; #P<0.05 vs. inhibitor NC. (C) MDA-MB-231 cells of untreated or transfected with mimics NC, mimics miR-301a-3p, inhibitor NC and inhibitor miR-301a-3p were subjected to flow cytometry analysis of apoptosis. (D) Quantification of apoptosis as shown in C. *P<0.05 vs. NC. miR, microRNA; NC, negative control.

miR-301a-3p suppresses cell apoptosis

The regulatory role of miR-301a-3p on apoptosis was subsequently determined in MDA-MB-231 cells using PI/Annexin V-FITC double staining and flow cytometric analysis. Compared with the cells transfected with the mimic NC, transfection with the miR-301a-3p mimic significantly inhibited the apoptosis of MDA-MB-231 cells. Conversely, miR-301a-3p knockdown induced MDA-MB-231 cell apoptosis (Fig. 2C and D). These results suggested that miR-301a-3p may suppress apoptosis in TNBC cells.

miR-301a-3p induces the migration and invasion of MDA-MB-231 cells

TNBC is a highly metastatic cancer. Thus, transwell plates with or without Matrigel precoating and wound healing assays were performed to analyze cell invasion and migration, respectively. No statistically significant differences were observed among the untreated, mimic NC-transfected and inhibitor NC-transfected MDA-MB-241 cells. The transfection of MDA-MB-231 cells with the miR-301a-3p mimic increased the migratory and invasive abilities. By contrast, transfection of cells with the miR-301a-3p inhibitor inhibited cell migration and invasion (Fig. 3). These data indicated that miR-301a-3p may function as an oncomiR in TNBC.

Figure 3 miR-301a-3p promotes the migration and invasion of MDA-MB-231 cells. (A) MDA-MB-231 cells, untreated (blank) or transfected with mimics NC, mimics miR-301a-3p, inhibitor NC and inhibitor miR-301a-3p were subjected to Transwell migration (upper images) and invasion (lower images) assays (magnification, x200). (B) MDA-MB-231 cells, untreated (blank) or transfected with mimics NC, mimics miR-301a-3p, inhibitor NC and inhibitor miR-301a-3p were subjected to wound healing assay. Images were captured at 0 h (upper panels) and 24 h (lower panels) (magnification, x200). (C and D) Quantification of cell migration and invasion as shown in A and B. **P<0.01. miR, microRNA; NC, negative control.

miR-301a-3p downregulates the expression levels of MEOX2

Downstream target genes of miR-301a-3p in MDA-MB-231 cells were subsequently identified. RT-qPCR analysis revealed that MEOX2 mRNA expression levels were downregulated in MDA-MB-231 cells following transfection with the miR-301a-3p mimic. Conversely, transfection with the miR-301a-3p inhibitor upregulated the expression levels of MEOX2 in MDA-MB-231 cells (Fig. 4A). The results of western blotting demonstrated that MEOX2 protein expression levels were also decreased and increased following the overexpression and knockdown of miR-301a-3p, respectively (Fig. 4B). Analysis of data from TCGA database revealed that miR-301a-3p expression levels were inversely associated with MEOX2 expression levels in breast cancer tissues (Fig. 4C). To investigate whether miR-301a-3p bound to the 3'-UTR sequence of MEOX2, WT and MUT 3'-UTR sequences of MEOX2 were synthesized and cloned into luciferase reporter vectors. The relative luciferase activity was subsequently measured following the overexpression of miR-301a-3p. The results demonstrated that miR-301a-3p overexpression reduced the relative luciferase activity of MDA-MB-231 cells when co-transfected with the WT, but not the MUT, 3'-UTR sequence of MEOX2 (Fig. 4D and E). Collectively, these results suggested that miR-301a-3p may directly target MEOX2 by binding to its 3'-UTR sequence.

Figure 4 MEOX2 is a downstream target of miR-301a-3p in MDA-MB-231 cells. (A) Western blot result of MEOX2 in MDA-MB-231 cells, untreated (blank) or transfected with mimics NC, mimics miR-301a-3p, inhibitor NC and inhibitor miR-301a-3p. (B) Relative protein level of MEOX2 in MDA-MB-231 cells, untreated or transfected with mimics NC, mimics miR-301a-3p, inhibitor NC and inhibitor miR-301a-3p. **P<0.01 vs. mimics NC; and ***P<0.001 vs. inhibitor NC. (C) Analysis of the correlation between miR-301a-3p and MEOX2 in breast cancer tissues from The Cancer Genome Atlas database. (D) WT or MUT sequence of MEOX2 3'-UTR was inserted into luciferase reporter vectors. At 48 h after transfecting MEOX2 overexpression vectors, luciferase activity was examined as indicated. **P<0.01 vs. control. (E) Binding sequence of miR-301a-3p with the 3'-UTR of MEOX2. MEOX2, mesenchyme homeobox 2; miR, microRNA; NC, negative control; WT, wild-type; MUT, mutant; UTR, untranslated region.

MEOX2 acts as a tumor suppressor gene in TNBC

Finally, the role of MEOX2 in TNBC cells was investigated. MEOX2 expression was knocked down by transfection with siRNA and overexpressed by the pcDNA3.1 vector RT-qPCR and western blotting confirmed successful MEOX2 knockdown and overexpression in MDA-MB-231 cells (Fig. 5A and B). The results of the MTT assay revealed that MEOX2 knockdown enhanced, while MEOX2 overexpression reduced MDA-MB-231 cell viability (Fig. 5C). Furthermore, MEOX2 was overexpressed in cells also overexpressing miR-301a-3p (Fig. 5D). The overexpression of MEOX2 further suppressed the viability of cells overexpressing miR-301a-3p (Fig. 5E). MEOX2 overexpression also increased the levels of apoptosis in MDA-MB-231 cells (Fig. 5F). These findings suggested that MEOX2 may serve as a tumor suppressor in TNBC.

Figure 5 MEOX2 suppresses MDA-MB-231 cell viability. (A) RT-qPCR and (B) western blot analyses of MEOX2 expression in MEOX2-knockdown and -overexpressing MDA-MB-231 cells. **P<0.01 vs. siCtrl and ***P<0.001 vs. Ctrl. (C) MTT assay was used to determine the viability of MEOX2-knockdown and -overexpressing MDA-MB-231 cells. *P<0.05 vs. mimics, **P<0.01 vs. siCtrl or vs. Ctrl, respectively. (D) RT-qPCR and western blot analyses of MEOX2 expression following transfection with mimics NC, mimics miR-301a-3p and mimics miR-301a-3p + MEOX2 in MDA-MB-231 cells. **P<0.01 and ***P<0.001 vs. mimics NC. (E) MTT assay was used to determine cell viability following transfection with mimics NC, mimics miR-301a-3p and mimics miR-301a-3p + MEOX2 in MDA-MB-231 cells. **P<0.01 vs. mimics NC; and #P<0.05 vs. mimics miR301a. (F) MDA-MB-231 cell apoptosis was analyzed following transfection with mimics NC, mimics miR-301a-3p and mimics miR-301a-3p + MEOX2. **P<0.01. MEOX2, mesenchyme homeobox 2; miR, microRNA; NC, negative control; si, small interfering RNA; Ctrl, control; RT-qPCR, reverse transcription-quantitative PCR.

Discussion

Breast cancer is the most common malignancy among women, and poses a major threat to women's health in both developed and developing countries. Over the past few decades, researchers have made significant efforts to determine the molecular mechanisms underlying breast cancer tumorigenesis. The expression of the ER has been reported as a major risk factor for breast cancer development. Tamoxifen, which interrupts the interaction between estrogen and the ER, is a drug approved for the treatment of ER+ breast cancer. The discovery of HER-targeted therapy also represented a significant milestone in breast cancer research. Trastuzumab, which was developed in 1990, inhibits the extracellular segment of HER2 and, combined with chemotherapy, can effectively prolong the overall and progression-free survival of patients with HER+ breast cancer (15-17). However, 15-20% of patients with breast cancer are diagnosed with TNBC, which remains difficult to treat as the currently available targeted therapies are not applicable, as this type of cancer is negative for ER, PR and HER2 expression. Therefore, there are currently no effective treatment options for patients with TNBC, except for traditional methods, such as surgical resection, radiotherapy and chemotherapy.

The present study aimed to determine the role of miR-301a-3p in TNBC. miR-301a-3p expression levels were upregulated in breast cancer tissues. Notably, the expression levels of miR-301a-3p were upregulated in TNBC samples compared with adjacent non-TNBC samples. The overexpression of miR-301a-3p increased the viability and migratory ability of MDA-MB-231 cells. By contrast, miR-301a-3p knockdown suppressed the viability and migration of MDA-MB-231 cells. Mechanistically, miR-301a-3p downregulated the expression levels of MEOX2 by directly binding to the 3'-UTR sequence of MEOX2. Further experiments demonstrated that MEOX2 acted as a tumor suppressor gene in TNBC.

miR-301a, which comprises miR-301a-3p and miR-301a-5p, was found to play a crucial role in cancer development, including pancreatic, colorectal and gastric cancer. The expression levels of miR-301a were reported to be upregulated in pancreatic cancer and promoted pancreatic cancer cell proliferation by targeting Bim (18). The knockdown of miR-301a-3p expression in pancreatic cancer cells was also found to increase the sensitivity of cells to gemcitabine treatment (19). In addition, miR-301a promoted the growth and invasion of colorectal cancer by targeting suppressor of cytokine signaling 6 or transforming growth factor β receptor 2 (20,21). In gastric cancer, miR-301a was also discovered to function as an oncogene. The overexpression of miR-301a was associated with a poor prognosis in gastric cancer and was found to downregulate RUNX family transcription factor 3 expression levels to promote disease progression (22,23). The role of miR-301a in breast cancer has also been previously investigated. For example, upregulated expression levels of miR-301a were found to be inversely associated with the prognosis of breast cancer (8,24). The overexpression of miR-301a also promoted breast cancer cell migration and invasion by downregulating the expression levels of PTEN, a tumor suppressor gene (9). The results of the present study revealed that the expression levels of miR-301a-3p were upregulated in TNBC tissues. MDA-MB-231 cells transfected with miR-301a-3p mimics exhibited increased viability compared with MDA-MB-231 cells transfected with NC mimics. The opposite effects were observed following miR-301a-3p knockdown. Furthermore, miR-301a-3p overexpression promoted MDA-MB-231 cell migration and invasion. These results suggested that miR-301a-3p may act as an oncogene in TNBC.

MEOX2 has been reported to function both as an oncogene and tumor suppressor, depending on the cancer type. For example, single-nucleotide polymorphism-based sequencing studies reported that MEOX2 served as a tumor suppressor gene in Wilms' tumor (25). In another study, MEOX2 expression was upregulated in laryngeal carcinoma and lung cancer tissues (26,27), and the overexpression of MEOX2 promoted laryngeal cancer growth by activating the PI3K/AKT signaling pathway. The upregulated expression levels of MEOX2 were also found to contribute to chemoresistance in lung cancer. In another study, MEOX2 was found to be downregulated by miR-301a in hepatocellular carcinoma (HCC) (28); miR-301a promoted HCC cell proliferation, migration and invasion by downregulating MEOX2 expression. However, to the best of our knowledge, the association between miR-301a-3p and MEOX2 in TNBC remains to be determined. The findings of the present study revealed that overexpression of miR-301a-3p downregulated MEOX2 expression levels in MDA-MB-231 cells. The results of the dual luciferase reporter assay demonstrated that miR-301a-3p directly bound to the 3'-UTR sequence of MEOX2. In addition, the knockdown of MEOX2 expression promoted TNBC cell viability. Further analysis also identified that the expression levels of miR-301a-3p were inversely correlated with those of MEOX2 in breast cancer samples. Thus, it was suggested that the reduction in MEOX2 expression may promote TNBC cell viability.

In conclusion, the results of the present study indicated that miR-301a-3p may serve as an oncogene in TNBC. The overexpression of miR-301a-3p promoted TNBC cell viability and migration, while the knockdown of miR-301a-3p exerted the opposite effects. Furthermore, miR-301a-3p was found to exert its effects in TNBC by downregulating the expression levels of MEOX2. The findings of the preset study highlight the importance of miR-301a-3p in TNBC and may indicate a possible target for the treatment of this disease.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by the Beijing Obstetrics and Gynecology Hospital, Capital Medical University (grant no. FCYY201816).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

HL and GW designed the current study. HL performed the experiments. GW supervised the present study. HL and GW confirm the authenticity of all the raw data. HL and GW drafted, reviewed and edited the manuscript. All authors read and approved the final version of manuscript.

Ethics approval and consent to participate

The protocol of the present study was approved by the Clinical Research Ethics Committee of Beijing Obstetrics and Gynecology Hospital, and written, informed consent was obtained from each patient for the use of their tissue prior to participation.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References