With the approval of the Ethics Committee of the First People’s Hospital of JingMen, we collected GC tissues (N = 30) and matched normal gastric tissues (N = 30) from GC patients during surgery in the First People’s Hospital of JingMen. The signed written informed consent was provided by the GC sufferers. All tissues were kept at -80˚C.
Procell (Wuhan, China) provided human AGS cells, HGC-27 cells, and GES1 cells. AGS cells were maintained in Ham’s F12K (Procell), and other types of cells were cultured in RPMI-1640 (Biosun, Shanghai, China) at 37˚C.
Vector establishment and cell transfection
GC cells at ~ 70% confluence were selected for cell transfection using TurboFect reagent (Thermo Fisher, Waltham, MA, USA) in accordance with the standard instructions. GenePharma (Shanghai, China) provided the small hairpin RNA targeting circ_0026359 (sh-circ_0026359, 5’-GATCCGTGATCCACGTGGTGCGGTTTCAAGAG AAACCGCACCACGTGGATCACTTTTTG-3’ and 5’-AATTCAAAAAGTGATCCACGTGGTGCGGTTTCACTTC AAACCGCACCACGTGGATCACG-3’), the mimics and inhibitors of miR-140-3p (miR-140-3p, 5’-UACCACAGGGUAGAACCACGG-3’ and anti-miR-140-3p, 5’-CCGUGGUUCUACCCUGUGGUA-3’), and the respective controls. The coding sequence of HOXA9 generated by polymerase chain reaction (PCR) as well as pcDNA 3.0 vector (pcDNA; Genomeditech; Shanghai, China) were used to build HOXA9 overexpression plasmid (HOXA9).
The extracted RNA using TsingZol (Tsingke, Shanghai, China) was employed for reverse transcription with EasyScript® cDNA Synthesis SuperMix (TransGen, Beijing, China) or miRNA reverse transcription reagents (TaKaRa, Dalian, China). An aliquot of 50–80 ng cDNA was reacted on a Bio-Rad 96-well Machine with qRT-PCR Mix (Tsingke) to perform a quantification analysis of circRNA/miRNA/mRNA. Finally, gene expression was analyzed using the 2−∆∆Ct method. The primer sequences are displayed in Table 1.
Identification of circ_0026359 location and stability
Circ_0026359 location was confirmed by the nucleocytoplasmic separation assay. In brief, the cultured GC cells were lysed on ice. Then, the PARIS™ Kit (Thermo Fisher) was used to extract nucleocytoplasmic RNA according to user’s manual. Expression of nucleocytoplasmic circ_0026359 was confirmed by qRT-PCR.
For identifying the stability of circ_0026359, 50 ng/mL Actinomycin D (Seebio Biotech, Shanghai, China) was incubated with AGS and HGC-27 cells for different time points. Then, RNA was isolated, and circ_0026359 and KRT7 levels were detected by qRT-PCR.
GC cells were plated in tissue culture microplates, and treated with sh-circ_0026359, miR-140-3p, anti-miR-140-3p or HOXA9. Forty-eight hours later, cell counting kit-8 reagent (Abcam, Cambridge, MA, USA) was added into each well and incubated for 2 h. The absorbance at 460 nm (A460) was measured using a microplate reader (Thermo Fisher).
Cell colony formation assay
GC cells stably expressing plasmids or oligonucleotides were diluted in media and placed in 6-well plates. About 2-week culture later, cell supernatant was removed. Paraformaldehyde (Seebio Biotech) was used to incubate the cells. After the cells were washed twice with deionized water, the number of colonies containing > 50 cells was determined.
Flow cytometry analysis
The cells were fixed with ethanol (Millipore, Bradford, MA, USA) overnight at 4˚C, and then harvested. Afterward, staining buffer, propidium iodide (Beyotime, Shanghai, China) and RNase A (Beyotime) were incubated with the cells. Flow cytometer was employed to assess DNA content and cell apoptotic rate.
Western blot analysis
Protein Extraction Kit (Phygene, Fuzhou, China) was used to prepare protein lysates. After measuring protein concentration, protein samples were added into each lane of polyacrylamide gels. After that, skimmed milk (Solarbio, Beijing, China) was utilized to block aspecific signals, and the membranes were incubated with anti-CyclinD1 (1:100; Abcam), anti-Bax (1:800; Otwo Biotech, Shenzhen, China), anti-HOXA9 (1:800; Otwo Biotech), β-actin (1:1000; Abcam), and secondary antibodies (1:10,000; Abcam). The protein blots were developed using eyoECL Plus.
Cells were suspended in FBS-free medium and dropwise added into the upper chambers coated with Matrigel (Corning, Madison, New York, USA). After 24 h later, the cells on the top surface of the lower chambers were treated with paraformaldehyde (Seebio Biotech) and crystal violet (Seebio Biotech), and photographed under a microscope (100 × ; Nikon, Tokyo, Japan). Cell migration assay was implemented in accordance with the above steps except that the upper chambers were not pre-coated with Matrigel.
Glucose consumption and lactate production
Cells were resuspended in Assay buffer (Abcam) and homogenized by pipetting up and down the tubes. Then, the supernatant was transferred to another tube, and endogenous LDH or enzymes were removed using Deproteinizing Sample Preparation Kit. The Reaction Mix or Background Reaction Mix was added into the test wells following the guidebook. Samples were assessed on microplate reader.
Mouse model assay
Under the agreement of the Animal Care and Use Committee of The First People’s Hospital of JingMen, the assay was implemented on 12 five-week-old male BALB/c nude mice (Charles River, Beijing, China). These mice were randomized into 2 groups: (1) sh-con group (injected with AGS cells stably expressing sh-con); (2) sh-circ_0026359 group (injected with AGS cells with circ_0026359 knockdown). The above cell suspensions containing 5 × 106 cells were injected into the upper back of nude mice. The volume (V) of peritoneal nodules was monitored once a week. On the 28th day, mice were anesthetized with xylazine, the forming tumors were harvested. Stable AGS cell line was established by GenePharma Co., Ltd.
Immunohistochemistry (IHC) assay
Ki-67 expression was detected by IHC assay according to the published methods . In short, the 4-µm-thick neoplasms from the mouse model assay were fixed, and dehydrated. The sections were heated at 80 °C and then incubated with anti-Ki-67 (1:80; Affinity, Nanjing, China) and secondary antibody (1:100; Affinity). Then, the samples were counterstained using hematoxylin (Phygene). The immunoreactivity was observed under a microscope.
Dual-luciferase reporter assay
The sequences of circ_0026359 and HOXA9 3’-untranslated region (3’UTR) containing the binding sites of miR-140-3p were amplified to establish the wild-type (WT) vectors (circ_0026359 WT and HOXA9 3’UTR WT). The mutant (MUT) plasmids including circ_0026359 MUT and HOXA9 3’UTR MUT were built by Yeasen (Shanghai, China). Then, the above vectors, miR-140-3p and miR-con were transfected into GC cells using TurboFect reagent (Thermo Fisher). Fluorescence signals were detected using a Dual-Lucy Assay Kit (Solarbio).
The results from 3 independent duplicate assays were expressed as means ± standard deviations. The significant differences between the 2 groups with Student’s t-tests or Wilcoxon rank-sum test, while among 3 or more groups with one-way analysis of variance. Log-rank test was used for comparing the disparities in overall survival curve, and a chi-square test was employed for comparing groups between low and high circ_0026359 expression. P value < 0.05 indicated statistical significance.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.