Patients and samples
Patients with TNBC (N = 33) were recruited from Taihe Hospital, Huibei University of Medicine. Tumor tissues and non-cancer tissues were surgically excised from patients with written informed consent. None of the patients had received any radiotherapy and chemotherapy before surgery. Samples were frozen in liquid nitrogen and preserved at -80℃ conditions. This study obtained the permission of the Ethics Committee of Taihe Hospital, Huibei University of Medicine.
All used cell lines were purchased from Bena culture collection (Beijing, China) and maintained at 37℃ conditions supplemented with 5% CO2. According to the instructions, MCF-10 A (non-cancer cells; control) and MDA-MB-231 cells were cultured in 90% DMEM (Bena) containing 10% FBS (Bena). MDA-MB-453 and MDA-MB-468 cells were cultured in Leibovitz’s L-15 medium (Bena) containing 10% FBS. BT-549 cells were cultured in RPMI1640 medium (Bena) containing 10% FBS.
Quantitative real-time polymerase chain reaction (qPCR)
Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from tissues and cells according to manufacturer’s protocol. RNA samples were reverse-transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Dalian, China) or using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Then cDNA was utilized for qPCR using the SYBR Green Master PCR mix (Applied Biosystems) on ABI 7900 system (Applied Biosystems). The expression levels were normalized by GAPDH or U6, using the 2−ΔΔCt method. All procedures were performed following the manufacturer’s instructions. The primers used were displayed in Table 1.
RNase R digestion
Partial isolated RNA samples were exposed to 1 µg RNase R (2 U/µg, Epicentre, Madison, WI, USA), followed by 1-h incubation at 37℃ conditions. Then, RNA samples were used for qPCR analysis.
The distribution of circ-CSNK1G1
The Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek Corp, Thorold, Canada) was applied to isolated cytoplasmic RNA and nuclear RNA, followed by the quantification of circ-CSNK1G1 using qPCR. GAPDH or U6 was used as the internal reference in the cytoplasmic fraction and nuclear fraction, respectively.
For circ-CSNK1G1 silence, small interference RNA (siRNA) specific to circ-CSNK1G1 (si-circ-CSNK1G1: 5’-TTCGAAATCAGGTGAAGGT-3’) and siRNA negative control (si-NC) were synthesized by Geneseed (Guangzhou, China). For circ-CSNK1G1 overexpression, pCD-ciR-circ-CSNK1G1 recombinant vector (circ-CSNK1G1) and empty vector control were constructed by Geneseed. The mimics and inhibitors targeting miR-28-5p (miR-28-5p and anti-miR-28-5p) and their negative control (miR-NC and anti-miR-NC) were purchased from RIBOBIO (Guangzhou, China). For LDHA overexpression, pcDNA-LDHA recombinant vector (LDHA) and empty vector control were assembled by Genecreate (Wuhan, China). All transfections were performed using Lipofectamine 3000 (Invitrogen).
Cell proliferation was assessed using MTT assay. Cells with different transfection were planted into a 96-well plate (3 × 103 cells/well). After culturing for 24 h, 48 h, and 72 h, cells in each well were treated with 10 µL MTT reagent (Abcam, Cambridge, MA, USA) for 4 h. Dimethyl Sulfoxide (DMSO) was then added to dissolve formazan crystals. Finally, the absorbance values at 490 nm were measured by a microplate reader (Bio-Rad, Hercules, CA, USA).
Colony formation assay
Colony formation assay was also applied to assess cell proliferation. Cells with different transfection were planted into a 6-well plate (200 cells/well). Cells were next incubated at 37℃ conditions containing 5% CO2 for 12 days to induce colony growth. Finally, cell surface was washed with PBS, fixed with paraformaldehyde and stained with crystal violet (Beyotime, Shanghai, China).
Flow cytometry assay
Cell apoptosis was distinguished by flow cytometry assay. The FITC Annexin V Apoptosis Detection Kit (Invitrogen) was applied for apoptosis analysis in this study. In brief, cells with different transfection were collected after digestion at 48 h post-transfection. Cells were washed with PBS and then treated with 5 µL Annexin V-FITC buffer and 10 µL propidium iodide (PI) solution, for 15 min in the dark. The apoptotic cells (Annexin V-FITC + and PI+/-) were distinguished using a flow cytometer (BD Biosciences, San Jose, CA, USA).
Cell migration and cell invasion were determined using transwell assay. Cells with different transfection were collected and resuspended into serum-free culture medium. Cells in serum-free culture medium were placed into the upper of transwell chambers (Corning Incorporated, Corning, NY, USA) coated with or without Matrigel (Corning Incorporated) for invasion assay or migration assay, respectively. The bottom of transwell chambers was filled with culture medium added with 10% FBS. Through 24-h incubation, cells that migrated or invaded into the lower surface were fixed with paraformaldehyde and stained with crystal violet, followed by photograph using a microscope (× 100 magnification; Olympus, Tokyo, Japan).
2-DeoxyGlucose (2-DG) treatment
2-DG is an inhibitor of the glycolysis pathway. Cells with different transfection seeded into a 96-well plate were incubated with 50 nM 2-DG. After culturing for 24 h, 48 h, or 72 h, cell viability was checked using by a microplate reader (Bio-Rad).
Glycolysis energy metabolism was assessed according to glucose consumption, lactate production and the ration of ATP/ADP. These indexes could be detected using matched kits, including Glucose Assay kit (Abcam), Lactate Assay kit (Abcam) and ADP/ATP Ratio Assay kit (Abcam). Following the protocols, glucose consumption, lactate production and ATP/ADP ratios were investigated to monitor glycolysis progression.
Tumor formation in vivo
For stable circ-CSNK1G1 knockdown, short hairpin RNA (shRNA) targeting circ-CSNK1G1 was synthesized by Geneseed and assembled into a lentiviral vector. BT-549 cells were infected with lentiviral sh-circ-CSNK1G1 (5’-GAAATCAGGTGAAGGTCTCCCTTCAAGAGAGGGAGACCTTCACCTGATTTCTTTTTT-3’) or negative control (sh-NC) and used for tumor formation assay. The experimental mice (Balb/c, female, 6-7-week-old) were purchased from Charles River (Beijing, China). BT-549 cells infected with sh-circ-CSNK1G1 or sh-NC were subcutaneously injected into the groin of mice (n = 6 per group). Mice were kept for 8 days until tumor stable growth. Then, tumor volume (length×width2 × 0.5) was measured every 3 days. At 23 days post-injection, all mice with anesthesia were sacrificed to remove tumor tissues. Animal study obtained the permission of the Animal Care and Use Committee of Taihe Hospital, Huibei University of Medicine.
Immunohistochemical (IHC) assay
Paraffin-embedded tissue Sect. (4 µm thick) were prepared. Tissue sections were deparaffinized and dehydrated. After antigen retrieval, sections were incubated with the primary antibodies, including anti-Ki67 (ab92742; Abcam), anti-MMP2 (ab235167; Abcam) and anti-MMP9 (ab228402; Abcam). Tissue sections were next exposed to Goat Anti-Rabbit secondary antibody. The sections were stained with 3, 3’-diaminobenzidine (DAB; Abcam), and then images were taken using a light microscope (Olympus).
Dual-luciferase reporter assay
The binding sites between miR-28-5p and circ-CSNK1G1 or LDHA 3’UTR were analyzed by starbase v3.0 (http://starbase.sysu.edu.cn/). The wild-type (WT) and mutant (MUT) sequences of circ-CSNK1G1 or LDHA 3’UTR containing miR-28-5p binding sites were inserted into the downstream of dual-luciferase reporter vector PGL4 to forming recombinant vector, WT-circ-CSNK1G1, MUT-circ-CSNK1G1, LDHA 3’UTR-WT and LDHA 3’UTR-MUT. For luciferase assay, MDA-MB-231 and BT-549 cells were cotransfected with miR-28-5p or miR-NC and above mentioned recombinant vector, respectively. After incubating for 48 h, luciferase activity was detected using dual-luciferase reporter assay system (Promega, Madison, WI, USA).
The expression of LDHA protein was detected by western blot. Total proteins were extracted using RIPA lysis buffer (Beyotime) and separated by 12% SDS-PAGE. The separated proteins were transferred onto PVDF membranes, followed by blockage in skim milk. The membranes were incubated with the primary antibodies against LDHA (ab125683; Abcam) and GAPDH (ab9485; Abcam), followed by incubation with the secondary antibody (ab205718; Abcam). Finally, the enhanced chemiluminescence (ECL) (Beyotime) reagent was used to visualize the signal on the membrane.
Statistical analysis was performed using GraphPad Prism 7.0 (GraphPad, Inc., La Jolla, CA, USA). The data were presented as mean ± standard deviation (SD). Student’s t-test and analysis of variance (ANOVA) were applied for difference comparison between two groups and in multiple groups, respectively. The correlation curve was depicted according to Pearson correlation coefficient. The curve of overall survival was generated by Kaplan-Meier plot and analyzed by log-rank test. P < 0.05 was considered to be statistically significant. Experiments, including qPCR, MTT assay, colony formation, flow cytometry assay, transwell assay and western blot, were repeated at least three times.