Patients and specimens
Tumor samples and paratumor specimens were obtained from 56 paired CRC patients who underwent surgical resection at China-Japan Union Hospital of Jilin University and immediately stored at − 80 °C until further analysis. All patients did not receive any preoperative treatment and were diagnosed by histopathological examination. his study was permitted by the Ethics Committee of China-Japan Union Hospital of Jilin University and all subjects had signed the written informed consents.
Cell culture and transfection
Human CRC cell line HCT116 and SW620, and normal colon (FHC) cells were obtained from Shanghai Academy of Life Science (Shanghai, China) and grown in the
Dulbecco’s modifed Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco) with at 37 °C in a humidified atmosphere with 5% CO2. Lentiviral plasmids encoding circ-BANP-specific short hairpin RNA (shRNA) (sh-circ-BANP, 5′-UGC UAA
UGA CAU UUU CCA GGG-3′) and shRNA scramble control (sh-NC) were designed and produced by Invitrogen (Carlsbad, CA, USA). HCT116 and SW620 cells were
transfected with lentivirus LV-3 (pGLVH1/GFP + Puro) vector and selected with puromycin (2–3 μg/mL) for 2 weeks to obtain sh-circ or sh-NC stably expressed cell
lines. The small interfering RNA (siRNA) sequences targeting circ-BANP covalent closed junction (si-circ-BANP, 5′-UCA UUG UUG AGU AUU ACU GUA-3′), siRNA negative control (si-NC), pcDNA3.1-circ-BANP overexpression vector (circ-BANP), and pcDNA3.1 empty vector (pcDNA), pcDNA3.1-MAPK1 overexpression vector (MAPK1) were also were purchased from Invitrogen. The miR-874-3p mimics, miR-874-3p inhibitor, and their corresponding negative control (miR-NC and inhibitor NC) were produced by RIBOBIO (Guangzhou, China). HCT116 and SW620 cells were plated into six-well culture plates (1 × 105 cells/well) at a confluence of 50 to 60%, and then plasmid, mimics, inhibitor and negative
control were transfected into cells using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions.
Quantitative real-time polymerase chain reaction
(qRT-PCR)
Total RNA was isolated using TRIzol reagent (Invitrogen) with the standard procedure. Subsequently, extracted RNA was incubated with Rnase R (Epicentre, Madison, WI, USA), followed by interaction with RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA, USA). After that, total RNA was reversely transcribed into complementary DNA (cDNA) by using the Prime Script RT Master Mix (Takara, Dalian, China), and then qRT-PCR was carried out on the ABI7500 system using SYBR Green methods. he following thermocycling conditions were used for the qPCR: Initial denaturation at 95 °C for 5 min; 40 cycles of 95 °C for 10 s, and annealing at 60 °C for 30 s. A melt curve step from 65-95 °C was performed in increments of 0.5 °C per 5 s. Relative transcription expression was analyzed by 2−△△Ct method and normalized by glyceraldehyde 3-phosphate dehydrogenase (GADPH) or U6 small nuclear B noncoding RNA (U6). he specific primer sequences were presented as follow: circ-BANP: F 5′-CAG GAC GGT CAG CGT CGT -3′, R 5′-GGC ACA GCG TTG CTA ATG AC-3′; MAPK1: F 5′-ACC AAC CTC TCG TAC ATC GG-3′, R 5′-GGG CTG ATT TTC TTG ATA GC-3′; miR-874-3p: F 5′-GAA CTC CAC TGT AGC AGA GAT GGT -3′, R 5′-CAT TTT TTC CAC TCC TCT TCT CTC -3′; GADPH: F 5′-GAT ATT GTT GCC ATC AAT GAC-3′, R 5′-TTG ATT TTG GAG GGA TCT CG-3′; U6: F 5′-CTC GCT TCG GCA GCACA-3′, R 5′-ACG CTT CAC GAA TTT GCG T-3′.
Cells viability assay
Transfected HCT116 and SW620 (1 × 104 cells/well) were seeded on the wells of a 96-well plate and cultured in DMEM including 10% FBS for 72h. Then 10 μL cell
counting kit-8 (CCK-8) solution (Beyotime, Shanghai, China) was supplemented into per well and incubated for another 2 h. Finally, the optical density (OD) at 450 nm
was determined by a microplate reader in the indicated time to assess the cell viability.
Transwell assay
Transfected cells (4 × 105 ) with serum-free DMEM were seeded on the top chambers, which membranes were pre-coated with matrigel (BD Biosciences, San Jose, CA, USA). Then the lower chambers were filled with 500 μL serum-DMEM. After incubation for 24h, cells on the lower face of the membranes were fixed and stained.
Finally, invaded cells in 5 randomly selected fields were counted with a microscope.
Measurements of glucose and lactate levels
After transfection for 48h, cells were maintained in a 6-well plate. 24 h later, supernatants of cell culture media were collected to detect the levels of glucose and lactate using a glucose and lactate assay kit (Sigma, St Louis, MO, USA) according to the manufacturer’s protocol. Levels were assessed based on the standard curve and
normalized by the protein concentration of samples.
Measurement of extracellular acidiication rate (ECAR)
ECAR was determined according to the XF Glycolysis Stress Test protocol on a Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA,
USA). Transfected HCT116 and SW620 cells (8 × 103 cells/well) were plated in a glucose-free Seahorse Assay plates. Following baseline examinations, per well were
sequentially added with 25 mM glucose, 1 μM oligomycin, and 50 mM glucose analog 2-deoxyglucose (2-DG) at indicated time points. Finally, ECAR were analyzed using the Seahorse XFe24 Extracellular Flux Analyzer and normalized to total protein concentration on each well.
Western blot
Total protein was extracted using RIPA buffer containing a proteinase and phosphatase inhibitor cocktail. Then isolated protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, shifted onto a polyvinylidene luoride membranes, and blocked with 5% non-milk. Afterwards, the membranes were incubated with primary antibodies against hexokinase-2 (HK2) (1:2000, ab104836, Abcam, Cambridge, MA, USA), pyruvate kinase M2 (PKM2) (1:3000, ab137852,
Abcam), MAPK1 (1:1000, ab241580, Abcam), proliferating cell nuclear antigen (PCNA) (1:5000, ab29, Abcam), Cyclin D1 (1:10,000, ab134175, Abcam),N-cadherin
(N-cad) (1:5000, ab18203, Abcam), E-cadherin (E-cad) (1:5000, ab15148, Abcam), hypoxia inducible factor-1α (HIF-1α) (1:1000, ab1, Abcam), glucose transport protein
1(GLUT1) (1:10,000, ab115730, Abcam), c-Myc (1:1000, ab32072, Abcam), and GAPDH (1:10,000, ab181602, Abcam), followed by interaction with HRP-conjugated
secondary antibody (1:1000, ab9482, Abcam). Finally, protein bands were visualized using the chemiluminescence chromogenic substrate (Beyotime, Beijing, China).
Dual-luciferase reporter assay
The circ-BANP and MAPK1 3′ UTR containing the wild-type (WT) or mutant (MUT) binding sites of miR- 874-3p were cloned into the pmiR-RB-Report (Promega, Shanghai, China), respectively. Subsequently, HCT116 and SW620 cells (3 × 104 cells) were seeded in a 24-wellplate individually and co-transfected with 200 ng of WT or MUT pmiR-RB-Report-circ-BANP/MAPK1 3′ UTR with 10 nM miR-18b-5p mimics or miR-NC using Lipofectamine™ 2000 (Invitrogen). Finally, a dual luciferase assay kit (Promega) was used to analyze the luciferase activity as described earlier [21].
Xenograft experiments in vivo
Five-week-old BALB/c nude mice (N = 6) were obtained from Jinan Pengyue Animal Center (Jinan, China). This study was approved by the Animal Research Committee
of China-Japan Union Hospital of Jilin University and was undertaken according to the guidelines of the National Animal Care and Ethics Institution. HCT116 cells
(1 × 105 ) stably transfected with lentivirus containing sh-circ-BANP or sh-NC were subcutaneously injected into the flanks of the nude mice. After 7 days following
the inoculation, the tumor size was examined 3 days and the tumor volume was calculated. After 21 days, all mice were sacrificed, and tumor masses were weighed and harvested for further molecular analysis.
Statistical analysis
Data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Inc., San Diego, CA, USA). Significant diferences between different groups were analyzed using Student’s t-test or one-way analysis of variance (ANOVA). The correlation analysis was performed using Spearman’s correlation test. The P value less than 0.05 indicated statistically significant.