In this study, serum samples from healthy volunteers (n = 59), breast cancer patients without bone metastasis (n=56) and breast cancer patients with bone metastasis (n =19) were tested. All patients were treated at The First Affiliated Hospital of Sun Yat‑sen University (Guangzhou, China), and the study was approved by its Institutional Review Boards. The Ethics Committee of The First Affiliated Hospital of Sun Yat‑sen University approved the procedures. Written informed consent for the use of human materials was approved according to The First Affiliated Hospital of Sun Yat‑sen University’s ethical guidelines, and all experiments were performed in accordance with the relevant guidelines and regulations.
4.2. Cell culture and transfection
The breast cancer cell lines MDA-MB-231, MCF-7 and SK-BR3 were purchased from the Chinese Academy Sciences Cell Bank and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS; HyClone, Marlborough, MA, USA) at at 37°C in a humidified-atmosphere containing 5% CO2. The overexpression vectors(Roche, Switzerland) for circRNA DENND4C (High- DENND4C) and HOXA9 (High-HOXA9), the miR-145-5p mimic and inhibitor (Invitrogen, CA, USA) and the short harpin RNA (shRNA)(Bioss, Beijing, China) for circRNA DENND4C (KD- DENND4C) and the small interfering RNA (siRNA)(Sigma‑Aldrich; Merck KGaA) for HOXA9 (KD-HOXA9) were each pre-transfected into breast cancer cells. The above vectors were transfected into breast cancer cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
4.3. RNA isolation and qRT-PCR amplification
Total RNA was extracted using TRIzol® reagent (Thermo) and cDNA was obtained using the SuperScript® cDNA Synthesis Kit (Thermo) according to the manufacturer’s instructions. Next, cDNA was transferred to a 20-μl PCR mixture composed of 2× UltraSYBR Mixture (Guang zhou, China). The primers were as follows: circRNA DENND4C forward: 5′-GGGGCAGCAGTATTGTGAAA-3′ and reverse, 5′-AAGACTGTGTGCTCCCCATT-3′); miR-145-5p forward 5′- CTGATGGTGGAGAGCTCACA-3′ and reverse 5′-GTGCAGGGTCCGAGGT-3′; U6 forward: 5′-GACTATCATATGCTTACCGT-3′ and reverse: 5′-GGGCAGGAAGAGGGCCTAT-3′; HOXA9 forward: 5′-CTTACCCAAGCTTCACTCACC-3′ and reverse: 5′- AAGAGGCCTGGTGCTACTAC-3′; β-actin forward: 5′-TCATGAAGTGTGACGTGGACATC-3′ and reverse: 5′-CAGGAGGAGCAATGATCTTGATCT-3′. U6 or β-actin was used as an internal control to normalize target gene transcripts.
4.4. Luciferase reporter assay
The amplified miR‑145‑5p inhibitor sequence and miR‑NC(Thermo Fisher Scientific, Inc) were transfected into breast cancer cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.
To identify the binding sequences and uniform resource locator, a luciferase reporter assay was performed. The miR‑145‑5p inhibitor or miR‑NC and the pRL‑TK vector (Promega Corporation) carrying the mutant (mut) or wild‑type (wt) DENND4C and HOXA9 3' untranslated region (3' UTR) were co‑transfected into breast cancer cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Three days later, the cells were lysed with Dual‑Glo®Reagent (Promega Corporation), and luciferase activity was measured using a Dual‑Luciferase Reporter Assay System (Promega Corporation). The firefly luciferase activity was normalized to Renilla luciferase activity
4.5. RNA pull-down assay
RNA pull-down assays were performed to identify the binding sites between miR-145-5p and circRNA DENND4C and the 3′UTR regions of HOXA9 mRNA. The biotin-labeled circRNA DENND4C and HOXA9 probes(Invitrogen, USA) were designed and constructed. Then, the above-mentioned probe-streptavidin Dynabeads were incubated with breast cancer cell lysates at 37 °C for 12 hours. Finally, to assess miR-145-5p enrichment, real -time qPCR was performed according to the manufacturer's instructions .
4.6. Western blot analysis
Proteins extracted from cell lysates or cell culture supernatant were subjected to electrophoresis. After the proteins were transferred and blocked, the membranes were incubated with rabbit anti-β-actin (1:1000; ab8227, Abcam, Cambridge, UK), anti-HOXA9 (1:1000; EPR3655(2), ab140631) anti-OCT4(1:1000; EPR17980, ab200834) anti-SOX2 (1:1000; ab97959) anti-Ck 14(1:1000; EP1612Y, ab51054), and anti-Ck18(1:1000; EPR1626, ab133263) overnight at 4°C.
4.7. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
An MTT assay was performed to evaluate the role of the circRNA DENND4C/miR-145-5p/HOXA9 axis in breast cancer cells. MDA-MB-231, MCF-7 and SK-BR3 cells were stably transfected with NC or KD-circ RNA DENND4C or KD-DENND4C+miR145-5p inhibitor or KD-DENND4C+High-HOXA9 and were seeded into 96-well plates at a density of 5 × 103 cells/well, after which they were incubated for 24 hours. Then, 20 μl of MTT solution (5 mg/ml) was added into each well and incubated for an additional 4 hours. After 160 μl DMSO was added to each well, the optical density (OD) value of each well was measured at 450 nm in a spectrophotometer (Philips, China).
4.8. Migration and invasion assays
Migration assays were performed in 48-well Transwell® plates (8.0-μm pore size; Costar, Corning Incorporated, USA). After pre-transfection with the overexpression vectors for circRNA DENND4C (High- DENND4C), High-DENND4C +miR-145-5p mimics, High- DENND4C +KD-HOXA9(siRNA) and NC-vectors, breast cancer cells were seeded into the upper chamber of a Transwell system at a concentration of 6 × 103 cells/well. After 24 hours of incubation, cells that had migrated to the bottom side of the Transwell membrane inserts were stained with crystal violet.
Invasion assays were performed using a Transwell chamber coated with Matrigel® (Becton Dickinson, USA). Briefly, cells from different groups were added into the upper chamber. After the Transwell system was incubated for 24 hours, cells that invaded to the bottom side of the membrane were fixed and stained with crystal violet.
4.9. Wound-healing assay
To further assess cell invasion, a wound-healing assay was performed in 6-well plates for 24 hours. After pretransfection with the siRNA for circRNA DENND4C silencing (KD-DENND4C), KD-DENND4C+miR-145-5p inhibitor, KD- DENND4C+High-HOXA9 (overexpression vectors for HOXA9) or NC-vectors, artificial wounds were generated with a sterile 10-µl pipette tip. Images were obtained under 100× magnification.
4.10. Experimental murine models of breast carcinoma bone metastasis
BALB/c-nu/nu mice (6-10 weeks of ages, 60-100 g, purchased from The Guangdong Medical Laboratory Animal Center) were maintained in a 12:12 hour light/dark cycle at 23˚C and 50‑70% humidity. Animals experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of The First Affiliated Hospital of Sun Yat‑sen University (Guangzhou, China), and in all experiments, the mice were euthanized with 50 mg/kg 1% sodium pentobarbital according to IACUC guidelines. We made an incision along the right femur of each mouse. A surgical scalpel was used to drill a tiny hole in the cortex, and MDA-MB-231 cells transfected with the KD-DENND4C vector, KD-DENND4C+miR-145-5p inhibitor, KD- DENND4C+High-HOXA9 or NC-vectors were injected into the bone marrow through the hole at a density of 5×106 cells per mouse. Then, we repaired the hole with bone wax and sutured the patellar tendon and wound. Tumor growth was monitored daily. After the animals were euthanized, we obtained tumor tissue under aseptic conditions. Then, tumor tissues were cut into small pieces and were subjected to enzymatic dissociation (0.2% collagenase, Abcam; Cambridge, UK) at 37°C for 90 min. The cell suspension was filtered through a 40‑µm filter (Biosharp Life Sciences, China). After dissociation, the cells were cultured in DMEM (HyClone; USA) supplemented with 10% FBS (Gibco;CA, USA.)
4.11. Immunohistochemistry (IHC)
Tissues were fixed in 10% neutral-buffered formalin and then paraffin-embedded. Paraffin-embedded tissues were deparaffinized and rehydrated for further 3,3'-diaminobenzidine peroxidase (DAB)-base immunohistochemical staining. Then, after proteolytic digestion and peroxidase blocking, the slides were incubated with a Ki67 antibody (1:100, ab15580, Abcam, USA) overnight at 4℃.
4.12. Flow cytometry assay
To assess the stem cell phenotype conversion, the cells were resuspended in 200 μl PBS and incubated with anti-CD44-APC (4 μl/ ml) and anti-CD24-BV510 (6 μl/ml) (BD Bioscience, USA) at 4°C for 1 hour. After the cells were washed three times with PBS and suspended in 100 μl PBS, the analysis was performed using a Flow CytoFLEX(Beckman Coulter, USA).
Cell apoptosis was examined using an annexin V-FITC/PI apoptosis detection kit (BD Bioscience, USA) according to the manufacturer’s protocol. After the cells were incubated with annexin V-FITC and PI at room temperature for 60 min in the dark, a flow cytometerFlow (Beckman Coulter, USA) was used to measure percentage of apoptotic cells.
4.13. Statistical analysis
Statistical significance was determined byStudent's t‑test which was used for comparisons between two groups and one‑way analysis of variance followed by Tukey's post hoc test for comparisons between more than two groups; analyses were performed using the Prism software package version 5.03 (GraphPad Software, La Jolla, CA, USA). Each experiment was repeated at least three times. All data are expressed as the mean ± SEM. A difference was considered statistically significant at a level of P < 0.05.