Circular RNA contributes to gastric cancer by targeting Wnt family member 2B as a competing endogenous RNA

Figure 1 Expression of hsa_circular-RNA_102415 is upregulated in gastric cancer tissues and cell lines. A: Volcano plot of circular RNAs (circRNA) with differential expression between different control samples and malignant tumor samples was illustrated- log10 was represented by the vertical axis, and log2 was represented by the horizontal axis. If microRNA is upregulated, it was indicated in red; if it was downregulated, it was indicated in blue; if there is no significant expression difference, it was indicated in black; B: The expression levels of hsa_circRNA_102415 detected in different CTRL samples and malignant tumor samples using the GSE83521 dataset are shown; C: Used quantitative real-time polymerase chain reaction to demonstrate the expression levels of hsa_circRNA_102415 in different CTRL samples and malignant tumor samples; D: The relevant content of using quantitative real-time polymerase chain reaction to detect the expression levels of hsa_circRNA_102415 in human gastric mucosal cell lines and gastric cancer cell lines was illustrated; E: The low and high expression levels of hsa_circRNA-102415 correspond to the overall survival rate. Data were reported using mean ± SEM (n = 3). Compared to GES-1, ^bP < 0.01. ^cP < 0.001. CTRL: Control; circRNA: Circular RNA; GC: Gastric cancer.

Figure 2 Hsa_circular-RNA_102415 silencing reduces gastric cancer cell proliferation and invasion and promotes apoptosis. Transfect small interfering-hsa_circular-RNA_102415 and control in BGC823 cells and AGS cells for 24 hours. A: Analyzed the relationship between gastric cancer cells and small interfering-hsa_circular-RNA_102415 using quantitative real-time polymerase chain reaction; B and C: Studied and analyzed the proliferation of cells transfected with cell counting kit-8; D and E: Studied and analyzed the invasion ability of cells after transfection using Transwell method; F and G: Used flow cytometry to study and analyze the level of apoptosis after cell transfection; H and I: Studied and analyzed the percentage of different cell cycle stages after cell transfection using flow cytometry. Data were reported using mean ± SEM (n = 3). ^bP < 0.001. circRNA: Circular RNA; si-NC: Small interfering negative control; FITC: Fluorescein isothiocyanate; OD: Optical density.

Figure 3 Hsa_circular-RNA_102415 is a microRNA-4529-5p sponge. A: Predicted the binding sites of microRNA (miR)-4529-3p and hsa_circular-RNA_102415 using databases such as circBank; B: The relative luciferase activity was detected using luciferase reporter gene analysis; C: The induction of gene enrichment in AGS cells by negative control (NC) probe and biotinylated miR-497-5p was detected using quantitative real-time polymerase chain reaction (RT-qPCR). Regarding the biological probe NC compared to D; D: Measured RNA binding protein immunoprecipitation using normal mouse IgG, cell lysate, and other inputs. Used RT-qPCR to detect the relative expression levels of two genes in AGS cells; E: Used RT-qPCR to detect the overexpression efficiency of miR-4529-3p mimetics in AGS cells; F: Used RT-qPCR to detect the expression level of hsa_circular-RN_102451 in transfected AGS cells; G: Used RT-qPCR to detect the expression level of miR-497-5p in transfected AGS cells; H: Used RT-qPCR to detect the relative expression levels of miR-4529-5p in human gastric mucosal cell lines and gastric cancer cells. Compared to GES-1. Data were reported using mean ± SEM. n = 3. ^bP < 0.01. circRNA: Circular RNA; miR: MicroRNA; MUT: Mutant; WT: Wild-type; NC: Negative control; Ago2: Argonaute 2; si-NC: Small interfering negative control; pcDNA: Plasmid DNA.

Figure 4 Hsa_circular-RNA_102415 accelerates malignant behaviors of gastric cancer cells by targeted inhibition of microRNA-4529-5p. Transfected microRNA (miR)-RNA-4529-5p mimic and plasmid DNA (pcDNA)-hsa-ccircRNA_102415 into AG cells. Transfected small interferin-hsa_circular-RNA_102415 and control group into BGC823 and AGS cells, with transfection time of 24 hours. A: The cell proliferation level caused by pcDNA-hsa_circular-RNA_102415 and other transfected cells was detected using cell counting kit-8; B and C: The level of cell invasion caused by pcDNA-hsa_ircRNA_102415 after cell transfection was detected using the Transwell method; D: The proportion of different stages of the cell cycle caused by cell transfection of pcDNA-hsa_ircRNA_102415 was detected using flow cytometry; E and F: Detect the level of cell apoptosis caused by pcDNA-hsa_ircRNA_102415 and other transfected cells using flow cytometry. ^bP < 0.01. circRNA: Circular RNA; miR: MicroRNA; NC: Negative control; NC: Negative control; pcDNA: Plasmid DNA; FITC: Fluorescein isothiocyanate.

Figure 5 MicroRNA-4529-5p targets WNT2B in gastric cancer cells. A: The binding sites of microRNA (miR)-4529-3p and WNT2B were predicted using the Targeted scan database; B: The relative luciferase activity between the two was detected using a luciferase reporter; C: RNA immunoprecipitation was used to detect the binding relationship between the two; D and E: Si-hsa_circRNA_102415 was transfected into AGS cells, and the expression level of WNT2B protein in the cells was subsequently measured using Western blotting; F: Transfect plasmid DNA-hsa_ircRNA_102415 into AGS cells, and then use Western blotting to measure the expression level of WNT2B protein in the cells; G: The correlation between the two was evaluated using Pearson correlation test; H: Pearson correlation test was used to evaluate the relationship between the two. Data were reported using mean ± SEM. ^bP < 0.01. circRNA: Circular RNA; miR: MicroRNA; NC: Negative control; si-NC: Small interfering- negative control; pcDNA: Plasmid DNA; MUT: Mutant; WT: Wild-type; RIP: RNA immunoprecipitation.

Figure 6 Hsa_circRNA_102415 enhances gastric cancer malignancy via the microRNA-4529-5p/WNT2B axis. Transfect small interfering-WNT2B and microRNA (miR)-4529-5p inhibitors into AGS cells for 24 hours. A: Studied and analyzed the transfected AGS cells, and detected the expression of miR-4529-5p using quantitative real-time polymerase chain reaction (RT-qPCR); B: Studied and analyzed the transfected AGS cells, and detect the protein expression of WNT2B using Western blotting; C: Studied and analyzed the transfected AGS cells, and detect the cell proliferation level using cell counting kit-8 method; D and E: Conducted research and analysis on transfected AGS cells, and used Transwell assay to detect the level of cell invasion; F: Study and analyze the transfected AGS cells, and use flow cytometry to detect the proportion of cell cycle stages; G and H: Study and analyze the transfected AGS cells, and detect the level of cell apoptosis using flow cytometry. Data were reported using mean ± SEM. ^bP < 0.01. circRNA: Circular RNA; miR: MicroRNA; NC: Negative control; si-NC: Small interfering negative control; pcDNA: Plasmid DNA; RT-qPCR: Quantitative real-time polymerase chain reaction; FITC: Fluorescein isothiocyanate.