Circ_0003356 suppresses gastric cancer growth through targeting the miR-668-3p/SOCS3 axis

Figure 1 Circ_0003356 is expressed to low levels in gastric cancer tissues. A: CircRNAs in GSE184882 were analyzed and five with the most significant expression differences in gastric cancer (GC) were used for heat map analysis; B: Relative expression of circRNAs in tumor and normal tissues determined by quantitative real-time polymerase chain reaction (qRT-PCR); C: Relative expression of circ_0003356 in tumor and normal tissues determined by qRT-PCR; D: Kaplan-Meier analysis of the correlation between circ_0003356 expression and overall survival of GC patients; E: Relative expression of circ_0003356 in GES-1, HGC-27, and AGS cells determined by qRT-PCR. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. NC: Negative control; GC: Gastric cancer.

Figure 2 Upregulation of circ_0003356 promotes cell apoptosis and suppresses proliferation, migration, invasion, and epithelial-mesenchymal transition of AGS and HGC-27 cells. A: Relative expression of circ_0003356 in AGS and HGC-27 cells detected by quantitative real-time polymerase chain reaction; B: Cell viability in AGS and HGC-27 cells assessed by CCK-8 assay; C: Proliferation of AGS and HGC-27 cells assessed by EdU assays. D: Apoptosis of AGS and HGC-27 cells assessed by flow cytometry; E: Migration of AGS and HGC-27 cells detected by wound healing assays; F: Invasion of AGS and HGC-27 cells detected by transwell assay; G: Levels of epithelial-mesenchymal transition-related proteins (E-cadherin and N-cadherin) in AGS and HGC-27 cells determined using western blotting. ^bP < 0.01, ^cP < 0.001.

Figure 3 Circ_0003356 directly binds to miR-668-3p. A: Fluorescence in situ hybridization assays were used to detect the sub-cellular localization of circ_0003356 in AGS and HGC-27 cells; B: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circ_0003356 in the nucleus and cytoplasm; C: Binding sequences between circ_0003356 and miR-668-3p predicted by circinteractome; D: Interaction between circ_0003356 and miR-668-3p in AGS and HGC-27 cells validated by RIP-qRT-PCR; E: interaction between circ_0003356 and miR-668-3p in AGS and HGC-27 cells validated by RNA pull-down assays; F: Interaction between circ_0003356 and miR-668-3p in AGS and HGC-27 cells validated by dual-luciferase reporter assays; G: Relative expression of miR-668-3p detected by qRT-PCR in tumor tissues and normal tissues; H: Correlation between circ_0003356 and miR-668-3p in gastric cancer tissues analyzed by Pearson’s correlation analysis. ^cP < 0.001.

Figure 4 Suppression of miR-668-3p promotes cell apoptosis and impairs the proliferation, migration, invasion, and epithelial-mesenchymal transition of AGS and HGC-27 cells. A: Relative expression of miR-668-3p in GES-1, AGS, and HGC-27 cells detected by quantitative real-time polymerase chain reaction (qRT-PCR); B: Cell viability in AGS and HGC-27 cells assessed by CCK-8 assay; C: Proliferation of AGS and HGC-27 cells assessed by EdU assays; D: Apoptosis of AGS and HGC-27 cells assessed by flow cytometry; E: Relative migration of AGS and HGC-27 cells detected by wound healing assays; F: Invasion of AGS and HGC-27 cells detected by transwell assays; G: Levels of epithelial-mesenchymal transition-related proteins (E-cadherin and N-cadherin) in AGS and HGC-27 cells determined using western blotting. ^bP < 0.01, ^cP < 0.001.

Figure 5 SOCS3 is targeted by miR-668-3p. A: Binding sites between SOCS3 and miR-668-3p were predicted by Starbase v2.0 software; B: Dual-luciferase reporter assays were used to confirm the targeting relationship between SOCS3 and miR-668-3p in AGS and HGC-27 cells; C: Relative expression of SOCS3 in AGS and HGC-27 cells detected by western blotting following miR-668-3p overexpression; D: Relative protein expression of SOCS3 in AGS and HGC-27 cells detected by western blotting after miR-668-3p inhibition; E: Relative mRNA expression of SOCS3 was detected by quantitative real-time polymerase chain reaction in tumor tissues and normal tissues; F: Correlation between circ_0003356 and SOCS3 in gastric cancer (GC) tissues analyzed by Pearson’s correlation analysis; G: Correlation between miR-668-3p and SOCS3 in GC tissues analyzed by Pearson’s correlation analysis. ^bP < 0.01, ^cP < 0.001.

Figure 6 Circ_0003356 overexpression suppresses the malignant behavior of gastric cancer cells via sponging miR-668-3p to target SOCS3. A and B: Protein expression of SOCS3 in AGS and HGC-27 cells detected by western blot; C: Cell viability in AGS and HGC-27 cells detected by CCK-8 assay; D: Proliferation of AGS and HGC-27 cells assessed by EdU assay; E: Apoptosis of AGS and HGC-27 cells assessed by flow cytometry; F: Relative migration of AGS and HGC-27 cells detected by wound healing assay; G: Relative invasion of AGS and HGC-27 cells detected by transwell assay; H: Levels of epithelial-mesenchymal transition-related proteins (E-cadherin and N-cadherin) in AGS and HGC-27 cells determined by western blotting. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 7 Circ_0003356 upregulation impedes gastric cancer tumorigenesis in vivo. A: Representative images of tumor in xenograft mice; B: Average volumes of xenograft mice; C: Average tumor weights of xenograft mice; D: Expression of E-cadherin, N-cadherin, Ki67, and SOCS3 in tumor tissues of xenograft mice determined by immunohistochemical staining; E: TUNEL staining to detect cell apoptosis in tumor tissues of mice; F: Expression of circ_0003356 and miR-668-3p determined by quantitative real-time polymerase chain reaction in tumor tissues of mice. ^bP < 0.01, ^cP < 0.001.