BC cells and specimens
All the BC cells and normal mammary cells (MCF-10A) were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured according to the supplier’s instructions. Fresh BC tissues, adjacent noncancerous tissues and corresponding paraffin embedded cancer tissues (n=120) were obtained from Harbin Medical University Cancer Hospital between 2012 and 2013. All the patients rerolled in our study had complete clinicopathological information and patients who received neoadjuvant chemotherapy, radiotherapy, immunotherapy and those with recurrent tumors, bilateral BC or other previous tumors were excluded. Our study was approved by the Research Ethics Committee of Harbin Medical University and all the patients enrolled in our study had signed informed consent.
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from fresh BC tissues and cells with TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instruction. Prime Script RT Reagent Kit with gDNA Eraser (Takara, Kyoto, Japan) was used to synthesize the cDNA and qRT-PCR was conducted using SYBR Premix Ex Taq™ II (Takara, Kyoto, Japan) on a CFX96 Touch Detection System (Bio-Rad, USA). Primers used in our study were listed in the supplementary table S1. GAPDH , U6 and 18S tRNA were used as internal controls, and relative RNA abundances were calculated by the standard 2-△△CT method.
Western blotting
Western blotting assays were performed as previously study reported15. Briefly, indicated cells were lysed with RIPA (Beyotime, China) buffer containing PMSF (Beyotime, China). The lysate was collected and western blotting assays were then performed. Antibodies used were listed in the supplementary table S2.
Cell proliferation assay
For the CCK-8 assays, indicated cells were seeded into 96-well plates at a density of 2×103 cells per well. At each pre-set time, 10μl CCK-8 reagent (Beyotime, China) were added into each well containing 90 μl culture medium and the plates were incubated at 37 °C for 2 hours. After that, the cell viability was detected by measuring the absorbance at 570 nm.
For the colony formation assays, indicated cells were seeded into six-well plates (5×102 cells/well) and incubated with culture medium containing 10% FBS at 37 °C for 2 weeks. After that, the cells were fixed with formalin for 30 minutes and stained with crystal violet. FluorChem M system was used to take the photos.
Edu staining assays were performed as follows: indicated cells were seeded into 96-well plates and cultured with 10% FBS medium at 37 °C. After the confluence reached to 60%-70%, the cells were subjected to Edu staining (Beyotime, China) according to the manufacture’s instruction. After fixing and permeabilizing, the cells were stained with Edu and DAPI solutions. The results were captured and analyzed under a fluorescence microscope.
Scratch assays and cell invasion assays
For wound scratch assays, indicated cells were seeded into six-well plates and cultured at 37°C. After the cells reached to 95% confluence, sterile micropipette tips were used to made the scratch wounds. Then the cells were washed by PBS and cultured with medium containing 10% FBS continuously. Images were taken in schedule under a microscope at preset time and the migration rate were subsequently analyzed.
For cell invasion assays, the upper champers of 24-well transwell plates (Coring, USA) were coated with Matrigel and incubated at 37℃ for 4 hours. Then 200μl serum-free medium containing indicated cells were added into the upper champers of the transwell plates and 600 μl 10% FBS medium were added into the lower champers of the transwell plates. Then the cells were cultured at 37℃ for 24 hours. Afterwards, the non-invaded cells were scraped from the upper side of the champers and invaded cells at the under-side were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Finally, the images were captured and the invaded cells were counted under a microscope.
Hematoxylin–eosin (HE) staining and immunohistochemistry (IHC)
HE staining assays were performed according to a standard HE staining technique as previously study reported16. Briefly, mouse lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned (5-mm thickness). After that, the lung sections were subjected to HE staining to evaluate pathological changes and photographed under an optical microscope.
IHC staining were performed on paraffin-embedded specimens from BC patients in a standard streptavidin-peroxidase complex manner. The staining results were evaluated with histochemistry score (H-score) by three senior pathologists independently. The following antibodies were used: UBE2O (Catalogue Number: 15812-1-AP, Proteintech Group, China), Ki-67 (Catalogue Number: 9449, CST, USA).
Sphere culture and sphere formation assays
For sphere formation assays, indicated cells (1×103) were trypsinized, resuspended with stem cell medium consisting of DMEM/F-12 (Catalogue Number: 12660012, Gibco, USA), 1×B27 (Catalogue Number: 17504044, Invitrogen, USA), 20ng/ml epidermal growth factor (Catalogue Number:PHG0311, Invitrogen, USA), 20ng/ml basic fibroblast growth factor (Catalogue Number: PHG0263, Invitrogen, USA), and 2mM L-glutamine (Catalogue Number: 25030081, Invitrogen, USA) and seeded into 24-well ultra-low attachment plates. Then the cells were cultured at 37℃ and the medium was refreshed every 48 hours. Two weeks later, the stemness spheres were observed and counted under an optical microscope.
Lentiviral production
Recombinant lentivirus for overexpression HCG18 or UBE2O in BC cells and corresponding negative controls were designed and synthesized from GenePharma (Shanghai, China). The recombinant lentivirus was transfected into indicated cells with polybrene (8μg/ml). Twenty-four hours after transfection, the cells were selected with puromycin and qRT-PCR was applied to detect the transfection efficiency.
To establish stable HCG18 or UBE2O knocking-down BC cells, shRNAs targeting HCG18 or UBE2O were designed from Sigma and transfected into indicated cells according to the manufactory’s instruction. The shRNA sequences were listed in supplementary table3. qRT-PCR was used to detect the knocking-down efficiency.
Transfection of BC cells
Small interfering RNAs (siRNAs) targeting HIF-1α and its control group were designed from GenePharma. The microRNA mimics and inhibitor were synthesized by Sigma. For transfection, indicated cells were seeded into six-well plates (5×105 per well) and cultured at 37℃ overnight. Then the cells were transfected with corresponding victors using Lipofectamine 2000 transfection reagent (Invitrogen, USA) according to the manufacturer's instructions. Forty-eight hours later, the transfected cells were harvested for the follow-up experiments. The siRNA sequences were listed in supplementary information table3.
Subcellular fractionation
Nuclear and cytoplasmic RNA separation was conducted using PARIS™ Kit (Catalogue Number: AM1921, Invitrogen, USA) according to the manufacturer’s instruction. qRT-PCR was used to analyze the expression of HCG18 in different fragments of BC cells.
RNA pull-down (RIP) assays
RIP assays were performed with Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA). Briefly, indicated cells were collected and lysed with RIPA. Then the lysate was subjected to RIP with an Anti-AGO2 antibody (Abcam, USA) according to the manufacturer’s instruction. Finally, the samples were disposed with proteinase K (Merck, USA) and qRT-PCR was used to analyze the binding of HCG18 and miR-103a-3p in BC cells.
Dual-luciferase reporter assay
To investigate whether miR-103a-3p could directly bind to the 3’UTR region of HCG18 or UBE2O, dual-luciferase report assays were performed using a double luciferase assay system (Promega, USA). In brief, 3’UTR region of HCG18 (wild /mutant type) or UBE2O (wild /mutant type) luciferase reporter plasmids and miR-103a-3p mimics were co-transfected into HEK-293T cells. Then the cells were lysed and luciferase assays were conducted according to the manufacturer’s instruction. Firefly luciferase activity normalized to Renilla luciferase activity was used as an internal control.
To analyze whether HIF-1α could bond to the promotor region of HCG18, HCG18 promotor region (wild /mutant type) luciferase reporter plasmids and HIF-1α plasmids were co-transfected into HEK-293T cells. Forty-eight hours later, cells were harvested and dual-luciferase assays were performed. Firefly luciferase activity normalized to Renilla luciferase activity was used as an internal control.
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed as previously study reported15. Briefly, indicated cells were treated with formaldehyde to crosslink target protein and chromatin and sonicated to an average length of 200-500bp on ice. Then a DNA depuration kit (Beyotime, China) was used to extract and clean the DNA fragments. After that, ChIP assays were conducted with an anti-HIF-1α antibody (Catalogue Number: 36169S, CST, USA) or IgG control. qRT-PCR was used to detect the promotor fragments of HCG18. The primers used in our experiment were listed in supplementary information.
Animal study
All the animal studies were approved by the Medical Experimental Animal Care Commission of Harbin Medical University and performed in Second Affiliated Hospital of the Harbin Medical University Laboratory. Animals used in our study were raised in specific pathogen-free animal facilities (Temperature was maintained at 25℃ and a12 hour light–dark cycle) and provided free access to clean water and food. Animals were anesthetized with 1–3% isoflurane and humanely sacrificed by CO2 inhalation as previous studies reported17. For the tumorigenesis assay, six-week-old BALB/c Nude mouse (Vitalriver, Beijing, China) were randomly assigned into two groups (n=6), and then MDA-MB-231NC or MDA-MB-231HCG18-KD (5×105) were respectively injected into the mammary fat pads of mice. The tumor growth was measured every week and seven weeks after injection, the mice were humanely sacrificed and the tumors were extracted and fixed in formalin solution. The tumor volumes were evaluated by the following formula: 1/2 (length × width2). For the lung metastasis model, MDA-MB-231NC or MDA-MB-231HCG18-KD (5×105) were respectively injected into the tail vein of the six-week-old BALB/c Nude mouse. Seven weeks after injection, the mice were humanely sacrificed and the lungs were collected to count metastatic nodules and for HE staining.
Statistical analysis
All the data were presented as the means ± SDs from at least three independent experiments. Student’s t-test or one-way ANOVA test were used to analyze the differences between groups. The chi-square test was used to analyze the relationship between HCG18 expression and the clinicopathological features of the BC patients. The Kaplan–Meier method and log-rank test were applied to draw the survival curves. P < 0.05 indicated statistical significance, which was evaluated by GraphPad Prism 8.0 software.