Background
Breast cancer (BC) is the second most common cancer worldwide and is a major cause of female death [
1,
2]. Some classical biomarkers, such as CEA and CA 15-3, detected in serum or nipple discharge, are associated with the development of BC [
3,
4]. But their tumour specificity is unsatisfactory. Furthermore, studies show that several genetic variants are potentially associated with BC risk, which provides insights into new potential biomarkers for BC [
5,
6]. However, biomarkers for the diagnosis and prognosis of human BC remain limited [
7]. Hence, it is still important to understand the underlying molecular processes of BC to explore novel biomarkers for BC progression and therapeutic targets.
Circular ribonucleic acids (circRNAs) are a class of stable, conserved ribonucleic acid (RNA) molecules [
8,
9]. Recent studies have indicated that circRNAs encode small peptides [
10,
11]. Most studies have proven that circRNAs act as sponges of microRNAs (miRNAs) to further regulate the development of multiple human diseases [
8,
12‐
16]. In human BC, many circRNAs have been discovered and proven to be related to the development of BC [
17]. Notably, a meta-analysis indicated that circRNAs have diagnostic value and may be potential diagnostic biomarkers for BC [
18]. However, research on the mechanisms of circRNAs in BC progression remains limited. In our previous study, a novel circRNA, hsa_circ_0043278, was identified and found to be downregulated in BC tissues [
19]. In addition, we predicted five putative target miRNAs. However, the underlying mechanisms of hsa_circ_0043278 remain to be elucidated. Thus, these findings prompted our interest in revealing the association between hsa_circ_0043278 and BC development.
In the present study, the low expression of hsa_circ_0043278 in BC cell lines was examined. Moreover, its target miRNA, miR-455-3p, has been considered an oncogene in BC owing to its inhibitory effect on etoposide-induced gene 24 (EI24) expression [
20]. EI24 has also been reported to have a role in suppressing tumour progression by inhibiting NF-κB activity [
21]. Hence, based on the aforementioned information, we hypothesized that hsa_circ_0043278 might influence BC development by targeting miR-455-3p and regulating EI24. Collectively, our results showed that hsa_circ_0043278 might act as a tumour suppressor gene in BC progression. Exploration of its molecular mechanism could be valuable for BC diagnosis and therapy.
Materials and methods
Tissue specimens
A total of 50 pairs of BC tissues and matched normal tissues were collected from BC patients at The First Affiliated Hospital of China Medical University (from January 2005 to December 2012). All patients were diagnosed with primary BC, female, between 27 and 81 years old, and did not receive chemotherapy or radiotherapy before surgery. Tissue specimens were obtained after surgical resection and stored at -80 °C in an ultralow-temperature freezer (Haier, China). The matched normal tissues were collected from regions more than 5 cm outside the edge of BC tissues. The study was approved by the Ethics Committee of The First Affiliated Hospital of China Medical University. All procedures performed in this study involving specimen collection and experiments were in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients.
Cell culture
Human BC cell lines (MDA-MB-231 and MCF-7) and a normal mammary epithelial cell line (MCF-10A) were purchased from the Cell Bank of the Chinese Academy of Sciences. The MDA-MB-468, BT-549, SK-BR-3, T47D and HEK 293T cell lines were maintained in our laboratory. MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% foetal bovine serum (FBS; HyClone, Logan, UT, USA). MCF-7 cells were cultured in minimum essential medium (MEM; Gibco) containing 10% FBS and 0.01 mg/ml insulin from bovine pancreas (Aladdin, Shanghai, China). Michigan Cancer Foundation-10A (MCF-10A) cells were cultured in mammary epithelial basal medium (MEBM; iCell Bioscience, Shanghai, China). The other cell lines were cultured appropriately. All these cell lines were maintained at 37 °C in a humidified incubator with 5% carbon dioxide (CO2).
qRT–PCR
Total RNA from tissues or cells was extracted with a simple Total RNA Kit (BioTeke, Beijing, China) following the manufacturer’s instructions. The purity and concentration of the RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Complementary deoxyribonucleic acid (cDNA) was reverse transcribed from the total RNA using Moloney Murine Leukaemia Virus (M-MLV) Reverse Transcriptase (BioTeke). Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) was performed in an Exicycler
TM 96 RT-PCR instrument (BIONEER, Daejeon, Korea) using the SYBR Green method (Takara Bio, Dalian, China). β-Actin was used as the control for the normalization of circRNA and messenger RNA (mRNA) levels, while U6 was used for normalization of miRNA levels. Table
S1 shows the primers for gene amplification, which were designed with Primer Premier 5 (PREMIER Biosoft, USA) and synthesized by Sangon Biotech (Shanghai, China). The relative expression of genes was analysed using the 2
-△CT method. All the experiments were repeated three times.
Vector construction and cell transfection
To overexpress hsa_circ_0043278, the mature sequence of hsa_circ_0043278 (chr17, 35797838–35800763) was synthesized and then cloned into the pCD5-ciR vector (GenScript, Nanjing, China) and was named “ov-circ”. A mock vector without the hsa_circ_0043278 sequence (ov-NC) served as the negative control. To knock down hsa_circ_0043278, two small interfering RNAs (siRNAs) targeting the back-splice junction site of hsa_circ_0043278 and a negative control siRNA (si-NC) were synthesized (GenScript). Consequently, qRT–PCR proved that siRNA-2 was the most effective siRNA, and it was used to construct the siRNA plasmid (Fig.
S1). The shRNA against hsa_circ_0043278 and the negative control shRNA were synthesized and cloned into the pRNAH1.1 vector, and the resulting vectors were named “sh-circ” and “sh-NC”, respectively. The sequences of the vectors and siRNAs were designed with the online NCBI database (
https://www.ncbi.nlm.nih.gov/). The miR-455-3p mimics and inhibitors were purchased from GenePharma (Shanghai, China). Vectors, mimics, and inhibitors were transiently transfected into BC cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocols. The sequences of the siRNAs and shRNAs are shown in Table
S2. The miR-455-3p inhibitor and negative control sequences are listed in Table
S3.
Western blotting
Proteins from tissues and cells were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Haimen, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF; Beyotime). A total of 40 μg (in 20 μl) of protein from each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and was then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Next, the membranes were incubated with 5% milk at room temperature for 1 h. Then, they were incubated with the appropriate primary antibody overnight at 4 °C and then with a horseradish peroxidase-conjugated secondary antibody (1:5,000; Wanleibio, Shenyang, China). The specific primary antibodies included anti-EI24 (1:1000, Proteintech, Wuhan, China) and anti-NF-κB (P65) (1:500, Wanleibio) antibodies. β-Actin and histone H3 (1:1000, Wanleibio) served as the internal controls. Immune complexes were finally visualized with an enhanced chemiluminescence system (Beyotime).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay
Cells were seeded in 96-well plates at a density of 3×103 cells per well. MTT solution (5 mg/mL; Sigma-Aldrich, St. Louis, Missouri) was added to each well at the indicated time points (0, 24, 48, and 72 h), and the plates were incubated at 37 °C in 5% CO2 for 4 h. Next, the medium was removed, and 200 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The optical density at 570 nm was recorded using an automatic microplate reader (BioTek, Vermont, USA).
Briefly, cells in each group were seeded in 35-mm dishes at 500 cells/dish and incubated at 37 °C in 5% CO2 for 2 weeks. The cells were fixed and stained with Giemsa solution (KeyGEN BioTECH, Jiangsu, China). Colonies consisting of at least 50 cells were counted and photographed under a microscope.
Wound healing assay
Cells were seeded (6×105 cells/well) and grown in 6-well plates for 48 h post transfection. Next, the cells were cultured in serum-free culture medium and treated with 1 μg/ml mitomycin C (Sigma-Aldrich) for 1 h. Wounds were created in the middle of the wells using 200-μL pipette tips. The cells were then washed and cultured in serum-free culture medium, and wound closure was monitored by imaging with a phase contrast microscope (Motic, Xiamen, China). After culture at 37 °C in 5% CO2 for 48 h, the widths of the wounds were measured again, and the migration rates were calculated in each group.
Migration and invasion assays
For the migration assays, 2×104 cells were seeded in the upper chambers of a Transwell plate (Corning Incorporated, Corning, NY, USA) in 200 μL of serum-free medium. An 800-μL volume of medium containing 20% FBS was added to the lower chambers. The pore size of the Transwell membrane was 8.0 μm. After incubation at 37 °C in 5% CO2 for 24 h, the cells that migrated to the bottom of the filter were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet (Amresco, USA) and were then photographed and counted under an inverted phase contrast microscope (Motic).
For the invasion assays, 2×104 cells in 200 μL of serum-free medium were seeded in the upper chambers of a Transwell plate with a Matrigel-coated membrane (BD Biosciences, NJ, USA). An 800-μL volume of medium containing 20% FBS was added to the lower chambers. The pore size of the Transwell membrane was 8.0 μm. After incubation at 37 °C in 5% CO2 for 24 h, the cells in the lower compartment were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet (Amresco) and were then photographed and counted under an inverted phase contrast microscope (Motic).
Xenograft experiment
A total of 18 two-month-old female BALB/c (nu/nu) mice were purchased from Wanleibio (Shenyang, China). The animal care and experimental procedures were approved by the Experimental Animal Ethics Committee of The First Affiliated Hospital of China Medical University. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH, Bethesda, Maryland, USA). We made great attempts to reduce the pain experienced by the animals. Mice were adaptively fed for 1 week under specific pathogen-free conditions. To establish xenograft tumours, 0.2 mL (5 × 107 cells/ml) of MDA-MB-231 cells stably transfected with the hsa_circ_0043278 overexpression vector or MCF-7 cells stably transfected with hsa_circ_0043278 shRNA was subcutaneously injected into the right axillae of the mice, and mice injected with mock vector or shRNA served as negative controls (n=3 mice in each group; 18 mice total). The tumour sizes were measured once a week with a calliper. The tumour volumes were calculated using the following formula: 1/2 (length × width2). In brief, mice were sacrificed in the 4th week, and the tumours were then excised, photographed and weighed.
Immunohistochemistry (IHC)
The expression of NF-κB (P65) in the paraffin-embedded xenograft tissue was detected by immunohistochemistry. Tissues were incubated with the anti-NF-κB (P65) (1:200; Wanleibio) primary antibody at 4 °C overnight and then with the secondary antibody at 37 °C for 60 min and an HRP-labelled streptavidin solution for 10 min. Next, tissues were stained by diaminobenzidine (DAB) (Solarbio, China) and observed under a microscope. The mean density of P65 in the tissues was calculated using Image-Pro software.
Dual-luciferase reporter assay
The sequences of wild-type hsa_circ_0043278 or a mutant without miR-455-3p binding sites were synthesized and subcloned into the luciferase reporter vector pmirGLO (GenScript); the resulting vectors were named “circ-WT” and “circ-Mut”, respectively. HEK 293T cells were cotransfected with the vectors and miR-455-3p mimics or negative controls. Relative luciferase activity was measured with a Dual-Luciferase Assay Kit (KeyGEN BioTECH) in accordance with the manufacturer’s protocols after 48 h of incubation. Luciferase activity was measured using a Tecan Infinite M200 Pro luminometer (Tecan, Männedorf, Switzerland).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA). The data for two groups were compared using a t test with the Wilcoxon signed-rank test. Comparisons of the means among multiple groups were performed by one-way analysis of variance (ANOVA) with the Kruskal–Wallis test. Pearson correlation coefficients were calculated for correlation analyses. Continuous data are presented as the mean ± standard deviation values (SD). A P value less than 0.05 was considered to indicate statistical significance. All experiments were repeated three times.
Discussion
BC represents the most common malignancy and the leading cause of cancer death in women worldwide [
24]. Thus, finding a new therapeutic target is an urgent need to improve the survival of BC patients. Sequencing technology has shown that circRNAs are involved in various malignancies and function as diagnostic markers or therapeutic targets [
8,
25]. circRNAs are recognized as a special class of RNA and are increasingly becoming a hotspot of RNA research. However, our knowledge of circRNAs is still limited [
25,
26]. The relationship between circRNAs and BC has rarely been reported. Discovering the biological roles of circRNAs could be beneficial for the diagnosis and treatment of BC.
Previously, we found that hsa_circ_0043278 was significantly downregulated in BC tissues. The downregulated expression of hsa_circ_0043278 in BC cell lines relative to normal mammary epithelial cells was verified. These findings led us to discover the correlation between hsa_circ_0043278 and BC. Thus, we selected MCF-7 (with the highest expression of hsa_circ_0043278) and MDA-MB-231 (with the lowest expression of hsa_circ_0043278) cells for subsequent research. Functional experiments suggested that overexpression of hsa_circ_0043278 significantly decreased the viability and the migration and invasion abilities of BC cells in vitro and decreased tumour growth in vivo, while downregulation of hsa_circ_0043278 showed the opposite effects. These results suggested that hsa_circ_0043278 might act as a tumour suppressor gene in BC development.
miRNAs have been reported to bind to the 3'UTR and inhibit the expression of their target mRNAs [
27‐
31]. CircRNAs have been generally reported to act as potent miRNA sponges [
12] via MREs, thus inhibiting miRNA expression [
32,
33]. In our previous study, we predicted that hsa_circ_0043278 contained 2 possible MREs for miR-455-3p [
19]. In addition, miR-455-3p has been reported to regulate EI24 expression in TNBC and to serve as an oncogene [
20]. Thus, we speculated that hsa_circ_0043278 might function by targeting miR-455-3p and regulating EI24 in BC. In our current study, miR-455-3p was found to be overexpressed in BC tissues and cell lines. The expression of miR-455-3p was negatively correlated with that of hsa_circ_0043278 in BC tissues. Moreover, the qRT-PCR and dual-luciferase reporter assay results suggested that hsa_circ_0043278 suppressed the expression of miR-455-3p by directly sponging it. The results proved the relationship between hsa_circ_0043278 and miR-455-3p in BC.
Furthermore, EI24 has been reported to be a target gene of miR-455-3p [
20]. It is also considered to act a tumour suppressor gene by inhibiting NF-κB activity [
21]. In the current study, EI24 and hsa_circ_0043278 were the most likely circRNA-miRNA target pair to share the same MRE for miR-455-3p. Next, we confirmed that EI24 was also markedly downregulated in BC tissues and cell lines. Our results showed that overexpression of hsa_circ_0043278 increased the EI24 level and suppressed the activity of NF-κB in BC cell lines. The rescue experiments demonstrated that inhibiting miR-455-3p partially suppressed the tumour-enhancing effect induced by silencing of hsa_circ_0043278 in MCF-7 cells. The changes in the level of EI24 and the activity of NF-κB were also partially reversed. Moreover, enhancing miR-455-3p expression showed the opposite effect in MDA-MB-231 cells. These results supported the hypothesis that hsa_circ_0043278 acts as a ceRNA of miR-455-3p to decrease the viability and inhibit the migration and invasion of BC cells. The hsa_circ_0043278/miR-455-3p/EI24 signalling axis might facilitate the progression of BC.
Additionally, we found that hsa_circ_0043278 expression was much lower in TNBC cell lines than in cell lines of other subtypes of BC. Thus, the relationship between TNBC and hsa_circ_0043278 needs to be verified in the future. The precise relationship between hsa_circ_0043278 and NF-κB also needs to be elucidated. Furthermore, the number of patient samples needs to be increased. Multicentre trials are also needed to investigate the roles of hsa_circ_0043278.
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