Background
Breast cancer is the most common malignancy with leading cause of death from cancer in women worldwide [
1]. Hypoxia is an important feature of solid tumors, which contributes to progression of breast cancer and increases the risk of metastasis and mortality [
2]. Under condition of hypoxia, glycolysis could maintain survival of cancer cells and promote cell progresses, such as proliferation, migration and invasion [
3]. Hence, it is urgent to explore new mechanism underlying breast cancer progression under hypoxia.
Circular RNAs (circRNAs) are a class of single-stranded noncoding RNA molecules, which play essential roles in development and diagnosis of human diseases and cancers [
4,
5]. Moreover, circRNAs have been regarded as potential therapeutic targets in breast cancer [
6]. Xu et al. reported that circRNA transcriptional adaptor 2A (circTADA2A) inhibits cell proliferation, migration and invasion by regulating microRNA-203a-3p/suppressor of cytokine signaling 3 (SOCS3) axis in breast cancer [
7]. Furthermore, Zhang et al. suggested that circRNA has_circ_0052112, generated from zinc finger 83, could promote cell migration and invasion by sponging microRNA-125a-5p in breast cancer [
8]. CircRNA differentially expressed in normal cells and neoplasia domain containing 4C (circDENND4C) is a hypoxia-associated RNA molecule [
9]. Notably, previous study indicated that high expression of circDENND4C promotes cell proliferation in breast cancer under hypoxia condition [
10]. However, the role and mechanism of circDENND4C in breast cancer progression remain largely unknown.
CircRNAs usually exhibit their biological roles by acting as microRNA (miRNA) inhibitors or sponges by binding the seed sites to quench the normal function of miRNA in human cancers [
11]. miRNAs are a class of small noncoding RNAs with 18–25 nucleotides, which play as promising targets for diagnosis, prognosis and therapeutics of breast cancer [
12]. Previous works have demonstrated that miR-200b and miR-200c could serve as important tumor suppressor by inhibiting cell proliferation, migration and invasion in breast cancer [
13‐
17]. More importantly, the complementary sites between circDENND4C and miR-200b or miR-200c predicted by bioinformatics analysis using starBase stimulated us to hypothesize that miR-200b and miR-200c might be required for circDENND4C-mediated progression of breast cancer.
In the current study, we measured the expression level of circDENND4C and investigated its biological role in glycolysis, migration and invasion in breast cancer cells under hypoxia. Moreover, we explored whether the regulatory mechanism was associated with miR-200b and miR-200c.
Materials and methods
Patients and tissues
A total of 43 patients with breast cancer were recruited from the Third Affiliated Hospital of Zhengzhou University. All participants have provided the written informed consents. The clinical features of patients were summarized in Table
1. The paired tumor tissues and corresponding adjacent normal samples were obtained prior to any other treatment and stored at -80 °C until used. The protocol of this study was approved by the ethics committee of the Third Affiliated Hospital of Zhengzhou University.
Table 1
Association between circDENND4C and the clinicopathological characteristics of breast cancer
Age(years) | P > 0.05 |
≥ 55 | 24 (55.8) | 13 (54.2) | 11 (45.8) | |
< 55 | 19 (44.2) | 10 (52.6) | 9 (47.4) | |
Menopause | P > 0.05 |
No | 26 (60.5) | 14 (53.8) | 12 (46.2) | |
Yes | 17 (39.5) | 9 (52.9) | 8 (47.1) | |
TNM stage | P < 0.05 |
I-II | 23 (53.5) | 8 (34.8) | 15 (65.2) | |
III-IV | 20 (46.5) | 15 (75.0) | 5 (25.0) | |
Lymph node metastasis | P < 0.05 |
No | 22 (51.2) | 8 (36.4) | 14 (63.6) | |
Yes | 21 (48.8) | 15 (71.4) | 6 (28.6) | |
Tumor size | P < 0.05 |
≥ 2 cm | 25 (58.1) | 17 (68.0) | 8 (32.0) | |
< 2 cm | 18 (41.9) | 6 (33.3) | 12 (66.7) | |
Subtype | | | | P > 0.05 |
TNBC | 19 (44.2) | 10 (52.6) | 9 (47.4) | |
HER2 | 5 (11.6) | 3 (60.0) | 2 (40.0) | |
Luminal A | 8 (18.6) | 4 (50) | 4 (50) | |
Luminal B | 11 (25.6) | 6 (54.5) | 5 (45.5) | |
Cell culture, hypoxia stimulation and cell transfection
Breast cancer cell lines (MDA-MB-453 and SK-BR-3) and normal human breast epithelial cells MCF-10A were purchased from the BeNa Culture Collection (Beijing, China), and cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) plus 10% fetal bovine serum at 37 °C with 5% CO2. For hypoxia stimulation, MDA-MB-453 and SK-BR-3 cells were growth in a hypoxia chamber with 1% O2 for various exposure times (0, 3, 6, 12, 24 and 48 h).
Small interfering RNA (siRNA) against circDENND4C (si-circ) (5′-AAGUAGCACUGCUCUUCAAAA-3′), siRNA negative control (si-NC) (5′-UCUCCGAACGUGUCACGUTT-3′), pcDNA-based circDENND4C overexpression vector (circ), pcDNA vector, miR-200b mimic (miR-200b) (5′-UAAUACUGCCUGGUAAUGAUGA-3′), miR-200c mimic (miR-200c) (5′-UAAUACUGCCGGGUAAUGAUGGA-3′), mimic negative control (miR-NC) (5′-UUCUCCGAACGUGUCACGUTT-3′), miR-200b inhibitor (anti-miR-200b) (5′-UCAUCAUUACCAGGCAGUAUUA-3′), miR-200c inhibitor (anti-miR-200c) (5′-UCCAUCAUUACCCGGCAGUAUUA-3′) and inhibitor negative control (anti-miR-NC) (5′-UUCUCCGAACGUGUCACGUTT-3′) were generated from Genepharma (Shanghai, China). When reaching 60% confluence, MDA-MB-453 and SK-BR-3 cells were transfected with 30 nM oligonucleotides or 200 ng vector using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufactures’ instructions. After transfection for 24 h, MDA-MB-453 and SK-BR-3 cells were collected for subsequent analyses.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Trizol reagent (Thermo Fisher Scientific, Waltham, MA USA) was used for RNA isolation from tissues or cells following the manufacturer’s protocols. For detecting level of circDENND4C, total RNA was treated by RNase R (Geneseed, Guangzhou, China) to improve purity of circRNA. The miScript Reverse Transcription Kit (Qiagen, Dusseldorf, Germany) was used for reverse transcription with 500 ng total RNA treated by RNase R or not and qRT-PCR was performed with the diluted cDNA products, special primers and SYBR Green mix (Thermo Fisher Scientific). The amplification conditions were: 95 °C for 1 min, 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The primers were as follows: circDENND4C (Forward, 5′-GGGGCAGCAGTATTGTGAAA-3′; Reverse, 5′-AAGACTGTGTGCTCCCCATT-3′); β-actin (Forward, 5′-TCATGAAGTGTGACGTGGACATC-3′; Reverse, 5′-CAGGAGGAGCAATGATCTTGATCT-3′); miR-200b (Forward, 5′-GCGGCTAATACTGCCTGGTAA-3′; Reverse, 5′- GTGCAGGGTCCGAGGT-3′); miR-200c (Forward, 5′-TAATACTGCCGGGTAATGATGGA-3′; Reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′); U6 (Forward, 5′-CTCGCTTCGGCAGCACA-3′; Reverse, 5′-AACGCTTCACGAATTTGCGT-3′). The relative expression levels of circDENND4C, miR-200b and miR-200c were determined with β-actin and U6 as internal control respectively by 2
-ΔΔCt method [
18].
Glucose consumption and lactate production
Transfected or non-transfected MDA-MB-453 and SK-BR-3 cells (1 × 105/well) were seeded into 6-well plates overnight and then incubated in hypoxia or normoxia condition for 48 h. Glucose Assay Kit and Lactate Assay Kit (Sigma, St. Louis, MO, USA) were used for detection of glucose consumption and lactate production respectively following the manufacturer’s protocol. The relative levels of glucose consumption and lactate production in all treated groups were normalized to normoxia group.
Western blot
After the indicated treatment, MDA-MB-453 and SK-BR-3 cells were collected and lysed in RIPA lysis buffer (Beyotime, Shanghai, China). The lysates were centrifuged at 12,000 g for 10 min and then total protein in supernatant was qualitied by using BCA protein assay kit (Beyotime). Following the boiled water bath, the equal amounts of proteins (30 μg) were loaded on SDS-PAGE gel and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking the non-specific binding sites with 5% non-fat milk, the membranes were probed with antibodies against hexokinase II (HK2) (ab227198, 1:5000, 102 kDa), matrix metallopeptidase 9 (MMP9) (ab38898, 1:1000, 92 kDa), MMP2 (ab97779, 1:1000, 72 kDa) or β-actin (ab8227, 1:3000, 42 kDa) overnight at 4 °C, along with horseradish peroxidase-labeled IgG (ab6721, 1:10000) for 2 h at room temperature. The antibodies used in this study were purchased from Abcam (Cambridge, MA, USA). The blots were visualized using BeyoECL Plus (Beyotime) and the relative protein level with β-actin as an endogenous control was normalized to normoxia group.
Trans-well assay
For migration and invasion assays, trans-well assay was performed using 24-well trans-well chamber pre-coated with or without Matrigel (BD Bioscience, San Jose, CA, USA). Treated MDA-MB-453 and SK-BR-3 cells (4 × 104/well) in serum-free RPMI-1640 medium were added to the upper chamber and 10% fetal bovine serum medium was added in the lower chamber. Cells were cultured at 37 °C with 5% CO2 for 24 h and then those on the lower surface were fixed with methanol (Sigma) and stained with 1% crystal violet (Sigma). A 200X magnification microscope (Olympus, Tokyo, Japan) was used to photograph the migrated and invasive cells with three randomly selected fields.
Bioinformatics analysis predicted the binding sites of circDENND4C and miR-200b or miR-200c by using starBase. The sequences of circDENND4C containing the predicted complementary sites of miR-200b or miR-200c (GCAGUAUU) at chr9: 1934268–1,934,275 were inserted into pmirGLO vectors (Promega, Madison, WI, USA) to generate wild-type luciferase reporter vector circDENND4C-WT (circ-WT). The corresponding mutant was generated by mutating the seed sites to AUGAGCAG, named as circDENND4C-MUT (circ-MUT). Luciferase assay was performed in MDA-MB-453 and SK-BR-3 cells co-transfected with 200 ng circ-WT or circ-MUT and 30 nM miR-200b, miR-200c or miR-NC using Lipofectamine 3000. A luciferase reporter assay kit (Promega) was used for the luciferase activity analysis at 24 h after the transfection.
For RIP assay, MDA-MB-453 and SK-BR-3 cells transfected with miR-200b, miR-200c or miR-NC were lysed in RIP lysis buffer. The combination of circDENND4C and miR-200b or miR-200c was examined by using the Magna RNA immunoprecipitation kit (Millipore) according to the manufacturer’s instructions. The magnetic beads were pre-coated by antibody against Ago2 (ab32381, Abcam) or IgG (AP112, Sigma). The level of circDENND4C (circ) enriched by RIP was measured by qRT-PCR.
Murine xenograft model
The lentiviral vectors with sh-circDENND4C (sh-circ) or corresponding control (sh-NC) were constructed by FulenGen (Guangzhou, China). MDA-MB-453 cells were infected with sh-circ or sh-NC for 6 h and then stably transfected cells were selected under fluorescence microscope (Olympus) and flow cytometry (BD Bioscience). Five-week-old female BALB/c nude mice (Vital River Laboratory Animal Technology, Beijing, China) were injected subcutaneously with stably transfected MDA-MB-453 cells (5 × 106), termed as sh-circ or sh-NC group (n = 3 per group). The mice injected with non-transfected cells were classified as empty group. Tumor volume was monitored every week and calculated using the formula: volume (mm3) = width2 × length/2. After 5 weeks following the inoculation, the mice were killed and tumor samples were weighted and harvested for measurement of circDENND4C, miR-200b and miR-200c expression levels. The experiment was permitted by the Animal Research Committee of the Third Affiliated Hospital of Zhengzhou University and performed in accordance with the guidelines of use of laboratory animals.
Statistical analysis
GraphPad Prism 7 software (GraphPad Inc., La Jolla, CA, USA) was used for statistical analyses. Data from three independent experiments were expressed as mean ± standard deviation (S.D.). The comparison between two or more groups was performed by using paired student’s t test or one-way ANOVA with Tukey’s post hoc test. The association between circDENND4C level and clinicopathologic features of breast cancer patients was analyzed by χ2 test. *P < 0.05, **P < 0.01 and ***P < 0.001 were considered significant.
Discussion
Hypoxia is a key feature of cancers and circRNAs are involved in regulation of hypoxia [
9]. Among all breast cancer-associated with circRNA, circDENND4C is a circRNA in response to hypoxia. This study was the first to investigate the effect of circDENND4C on glycolysis, migration and invasion in breast cancer and explore the potential sponging miRNAs.
To resist hypoxia stress, cancer cell would trigger hypoxia-inducible factor 1 alpha (HIF1A) expression, which is related with dysregulation of circRNA. Previous study suggested that circDENND4C is a HIF1A-associated circRNA, which is highly expressed in hypoxia condition and promotes cell proliferation at 4 days in breast cancer [
10]. Similarly, we also found that circDENND4C expression was enhanced in breast cancer cells after treatment of hypoxia. Moreover, high expression of circDENND4C predicted poor outcomes of patients with breast cancer. The hypoxia environment would lead to the reprogramming of glycolytic metabolism, which is characterized by glucose consumption and lactate production. Furthermore, HK2 is a key enzyme associated with glycolysis in cancers, including breast cancer [
19‐
21]. Under hypoxia, glucose consumption, lactate production and HK2 protein level were enhanced in breast cancer cells, indicating that glycolysis was triggered. However, loss-of-function experiments uncovered that circDENND4C knockdown could decrease glycolysis in breast cancer cells under hypoxia. Besides, hypoxia-induced epithelial mesenchymal transition is an important mechanism of tumor progression, which is responsible for cancer cell migration and invasion [
22,
23]. MMPs, especially MMP9 and MMP2, are crucial biomarkers for migration and invasion in breast cancer [
24‐
26]. By detecting MMPs levels and trans-well assay, we found that cell migration and invasion were increased in cells under hypoxia by increasing MMP2 and MMP9, which was inhibited by silence of circDENND4C. We hypothesized that anti-metastatic role of circDENND4C silence might be associated with epithelial mesenchymal transition, which needs further study in future. These indicated circDENND4C as a therapeutic target for breast cancer patients.
Prior studies have revealed that circRNAs could serve as sponges of miRNAs [
27‐
29]. To figure out whether circDENND4C regulates breast cancer progression by sponging miRNAs, its targets were explored by bioinformatics analysis using starBase, predicting that miR-200b and miR-200c might be sponged by circDENND4C, which was confirmed by luciferase activity and RIP assays. Our research showed that miR-200b and miR-200c expression levels were decreased in breast cancer cells after exposure of hypoxia, uncovering that they might be associated with regulation of hypoxia. Previous studies demonstrated that miR-200b and miR-200c could inhibit glycolysis in cancers by targeting lactate dehydrogenase A and sirtuin 2 [
30,
31]. Additionally, miR-200b and miR-200c could suppress migration and invasion of breast cancer cells by regulating ezrin-radixin-moesin and fucosyltransferase-4 [
32,
33]. These reports suggested the anti-cancer roles of miR-200b and miR-200c by decreasing glycolysis, migration and invasion in human cancers. The current study using rescue experiments revealed that knockdown of miR-200b and miR-200c attenuated the anti-cancer role of circDENND4C silence in breast cancer under hypoxia, indicating that circDENND4C regulates breast cancer progression by sponging miR-200b and miR-200c. Besides, in vivo experiments further supported the suppressive effect of circDENND4C interference by increasing miR-200b and miR-200c. The function of miRNAs is realized by regulating mRNA expression, hence the promising targets of miR-200b and miR-200c should be explored in further study. Besides, hypoxia is regulated not only by HIF1A, but also via mTORC1 signaling. Here we found that knockdown of circDENND4C suppressed the activation of p70S6K1, a marker of mTORC1 signaling (Additional file
11: Figure S11A and 11B), indicating the importance of this pathway for mechanism mediated by circDENND4C. While the more details need further study in future.
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