Patients and tissues
In this study, a total of 80 pairs of GC tissues and adjacent tissues were collected from patients who were diagnosed with GC at the First People's Hospital of Neijiang and the Affiliated Hospital of Guizhou Medical University. All patients signed an informed consent form before the operation and did not suffer from other malignant tumors or receive any preoperative chemotherapy or radiotherapy. The tissue samples after surgical were immediately snap-frozen in liquid nitrogen, and then transferred to -80℃ refrigerator for long-term storage for until used. In addition, the tissue were identified by two senior pathologists. At the same time, our research was also approved by the Ethics Committee of the First People's Hospital of Neijiang City and the Affiliated Hospital of Guizhou Medical University.
Cell culture and treatment
The gastric cancer cell lines HGC-27, MGC-803, BGC-823, SGC-7901, MKN45 and normal gastric epithelial cell line GES-1 were purchased from the Cell Center of Shanghai Institutes for Biological Sciences. The human gastric cell lines were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA), which supplemented with 10% FBS (Invitrogen) and 1% streptomycin (Gibco, USA). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. The cells are replaced with culture medium every other day or every day according to the state of the cells. When cells are passaged, 0.25% trypsin is used to digest the cells.
Plasmid construction and cell transfection
To silence circ-BVES expression, small interfering RNA (siRNA) against circ-BVES (sh-circ)(5’-ATGTCCTCTCTTCGTAAAGAT-3’)and(5’-AGCATGTCCTCTCTTCGTAAA-3’), miR-145-5p mimic (5’-GUCCAGUUUUCCCAGGAAUCCCU-3’), mimic negative control (miR-NC) (5’-UCACAGCCUCCUAGACAGACUAUA-3’), miR-145-5p inhibitor (inh-145-5p) (5’- CAGGTCAAAAGGGTCCTTAGGGA-3’) and negative control (inh-NC) (5’-UUCUCGGAUCGUGACACGUTT-3’) were generated from Geneseed (Guangzhou, China). When cells grow in log phase, SGC-7901 and MKN45 cells were transfected using Lipofectamine 3000 (Invitrogen; USA) following the manufactures’ instructions. After cultured for 24–48 h, SGC-7901 and MKN45 cells were collected for subsequent experiment.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolation from tissues or cells and reverse transcribed into cDNA using Prime Script RT Reagent Kit (Takara, Japan) following the manufacturer’s protocols. qRT-PCR was conducted on a Bio-Rad CFX96 system (Bio-Rad, CA, USA) with TB Green Premix Ex Taq (Takara, Japan). GAPDH and U6 were respectively selected as internal controls for circRNA and miRNA. The relative expression levels were calculated according to the 2−ΔΔCt method. The primers sequences were as follows: circBVES (Forward, 5’-TGGAACATCAGCTCAGCCTC-3’; Reverse, 5’- TCAATCTTTACGAAGAGAGGACA-3’);miR-145-5p(Forward,5’-GTCCAGTTTTCCCAGGAATCCCT-3’; Reverse,5’-CAGGTCAAAAGGGTCCTTAGGGA-3’); U6 (Forward,5’-CTCGCTTCGGCAGCACA-3’;Reverse,5’-AACGCTTCACGAATTTGCGT-3’).
Cell Counting Kit-8 (CCK-8) assay
The proliferation of GC cells was detected by CCK8 assay (Dojindo Laboratories, Kumamoto, Japan). Cells in the logarithmic growth were collected and seeded into 96-well plates at a concentration of 1 × 103 cells/ml. After culture for 24, 48, 72, and 96 h, 10 µl of CCK8 solution was added into each well and further cultured for another 2 h. Finally, the optical density of each hole was measured at a wavelength of 450 nm.
Detection Of Extracellular Acidification Rate (ECAR) And Cellular Oxygen Consumption Rate (OCR)
ECAR and OCR were detected by the Seahorse Bioscience XF96 Extracellular Flux Analyzer, following the manufacturer's instructions of Seahorse XF Cell Mito Stress Test Kit and Glycolysis Stress Test Kit (Seahorse Bioscience, Billerica, MA, USA). Cells were seeded in XF96 Cell Culture Microplates (Seahorse Bioscience) at a density of 1 × 104 cells/well. For ECAR detection, glucose, oligomycin, and 2-deoxyglucose were automatically added to each well. For OCR measurement, oligomycin, FCCP (Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) and rotenone were added into XF96 Cell Culture Microplates. The data of ECAR and OCR analyzed by the XF-96 wave software.
Glucose consumption, Adenosine Triphosphate (ATP) Synthesis and Lactate Production
The Glucose Assay kit, Lactate Assay Kit and ATP Assay kit (Invitrogen, USA) were used to determined the GC cells glucose consumption, Adenosine Triphosphate (ATP) Synthesis and Lactate Production. All measurements followed manufacturer's instructions and were normalized for the number of cells in each experiment.
5–8 weeks old male BALB/c mice were performed using in vivo tumor growth assays. In short, SGC-7901 cells stably transfected with empty vector or si-circBVES (JiKai, Shanghai, China) and selected with puromycin. Then the cells harvested and resuspended in culture medium (2 × 106cells/ml). For xenograft experiments, subcutaneously inoculated the cells in the concentration into female BALB/c mice. The tumor volume was measured once a week and calculated to be 0.5 × length × with2. The mice were sacrificed after 6 weeks, and tumor tissues were taken for further analysis.
Immunohistochemical (IHC) analysis
Mouse tumor tissues were fixed with 10% formalin, dehydrated, paraffin-embedded and cut into 4 µm specimens. Subsequently, the samples were dewaxed with xylene and rehydrated with graded alcohol. The specimens were repaired with sodium citrate, blocked with hydrogen peroxide, and bovine serum albumin. After incubating with Ki67 and PCNA at 4 °C overnight, the sections were washed twice and coupled with HRP-polymer at room temperature. After washing, 3,3'-diaminobenzidine (DAB) substrate (Sigma) was added to the tissue section, waiting for the color to change. The sections were counterstained with hematoxylin and observed through a microscope.
RNA-binding protein immunoprecipitation assay
All the steps of RNA binding protein immunoprecipitation (RIP) detection are performed following to the previous method according to the manufacturer's instructions of Magna RIP Kit (MilliPore). The interaction between miR-145-5p and circ-BVES in SGC-7901 and MKN45 cells was verified by biotin labeled miR-145-5p probes. The probe was designed and obtained from Sangon Biotech(Shanghai, China). The SGC-7901 and MKN45 cells were lysed with RIP lysis buffer, and the magnetic beads were incubated at 4 °C overnight. The enrichment of Circ-BVES was analyzed by RT-QPCR.
Dual-luciferase reporter assay
In order to confirm the interaction between circ-BVES and miR-145-5p, miR-145-5p and target gene HMGB3, we constructed circBVES-WT, circBVES-Mut, HMGB3 3'UTR-WT and HMGB3 3'UTR- Mut recombinant plasmid. All plasmids were confirmed by sequencing. The relative luciferase activity of the treated SGC-7901 and MKN45 cells were examined using the dual luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's protocol.
Western blot assay
Total protein was extracted from the treated SGC-7901 and MKN45 cells using a protein extraction kit (Key Gene,China) according to the manufacturer's instructions. The concentration of each sample was quantitated by BCA kit (Servicebio). The equivalent proteins of each group were separated by 10% SDS-PAGE under 90 V voltage according to molecular weight, and the separated proteins were transferred to PVDF membranes (microwells, USA). After blocking with 5% skim milk for 2 h, they were incubated with primary antibodies against CCNE1 (1:1000), CDK2 (1:1000), HMGB3 (1:1000) (Abcam, USA), GAPDH (1:1000) at 4 °C overnight. After washing and removing the free antibody with TBST buffer, incubate with the secondary antibody at 1:2000 dilution at room temperature for 2 h. Finally, the enhanced chemiluminescence (ECL) kit (Pierce, Waltham, MA, U.S.A.) was used to observe the immunoreactive bands.
All statistical analyses in the present study were performed using SPSS 17.0 and GraphPad Prism 7.0 software. Data were showed as mean ± standard deviation (SD). Comparisons between two groups were conducted using the twin tailed Student’s t-test. The correlation between groups was analyzed by Pearson correlation. P value < 0.05 was considered as statistically significant.