18β-glycyrrhetinic acid promotes gastric cancer cell autophagy and inhibits proliferation by regulating miR-328-3p/signal transducer and activator of transcription 3

Figure 1 Drug target miR-328-3p of 18β-glycyrrhetinic acid and its relationship with cell proliferation. A: Chemical structure of 18β-glycyrrhetinic acid (18β-GRA); B: Volcanic map; C: Heat map; D: Effect of 18β-GRA on expression of miR-328-3p in gastric cancer cells; E: Expression of miR-328-3p in gastric cancer cells transfected with lentivirus; F: Lentivirus transfected gastric cancer cells, cell status and transfection efficiency; G: Effect of overexpression of miR-328-3p on survival rate of AGS cells. All analyses were repeated three times. The data is represented as the mean ± SD. ^bP < 0.01; ^cP < 0.001. NC: Negative control; Vector: The blank vector for lentivirus transfection; GFP: Green fluorescent protein.

Figure 2 Effects of overexpression of miR-328-3p on clone formation ability, cell cycle and apoptosis of gastric cancer cells. A and B: Effect of miR-328-3p on colony formation ability of AGS cells; C and D: Effect of miR-328-3p on cell cycle of AGS cells; E and F: Effect of miR-328-3p on apoptosis of AGS cells. All analyses were repeated three times. The data is represented as the mean ± SD. ^bP < 0.01; ^cP < 0.001. Vector: The blank vector for lentivirus transfection.

Figure 3 Effect of miR-328-3p on subcutaneous tumor size in nude mice. A: Comparison of xenotransplantation models of gastric cancer in nude mice; B: Subcutaneous tumor transplantation in nude mice; C: Tumor growth curves of the transplanted tumor model; D: The influence of subcutaneous tumor formation on body weight of nude mice; E: Hematoxylin and eosin (HE) staining and immunohistochemical (IHC) staining of transplanted tumor tissue; F: Statistical graph of IHC staining results. All analyses were repeated three times. The data is represented as the mean ± SD. ^aP < 0.05; ^bP < 0.01. NC: Negative control; Vector: The blank vector for lentivirus transfection.

Figure 4 Signal transducer and activator of transcription 3 is targeted by miR-328-3p. A: TransmiR v2.0 database predicted miR-328-3p related proteins; B: STRING database predicted the relationship between Signal transducer and activator of transcription 3(STAT3) and autophagy-related proteins; C: Effect of miR-328-3p on the expression of STAT3 mRNA; D and E: Effect of miR-328-3p on the expression levels of STAT3 and phosphorylated STAT3; F: Schematic diagram of binding site between miR-328-3p and STAT3 3’ untranslated regions; G and H: STAT3 is the target gene of miR-328-3p. All analyses were repeated three times. The data is represented as the mean ± SD. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. NC: Negative control; Vector: The blank vector for lentivirus transfection; WT: Wild type; Mut: Mutant.

Figure 5 18β-glycyrrhetinic acid regulates miR-328-3p/signal transducer and activator of transcription 3 signaling pathway and promotes the synthesis of autophagosomes in gastric cancer cells. A and B: Effect of miR-328-3p on the expression level of light chain 3 II (LC3 II) in AGS cells; C and D: Effects of 18β-glycyrrhetinic acid (18β-GRA) on LC3 II expression in AGS cells; E and F: Monomeric red fluorescent protein-green fluorescent protein-LC3 tandem fluorescent protein adenovirus-infected AGS cells, and effects of different multiplicity of infection on cell status and infection efficiency; G and H: Effects of laser confocal imaging of 18β-GRA combined with bafilomycin A₁ (Baf A₁) on autophagy flow; I: Effect of 18β-GRA on the expression of signal transducer and activator of transcription 3 (STAT3) in AGS cells; J and K: Effects of 18β-GRA on the expression of STAT3 and phosphorylated STAT3 in AGS cells. All analyses were repeated three times. The data are represented as the mean ± standard deviation. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. NC: Negative control; Vector: The blank vector for lentivirus transfection.

Figure 6 Graphic abstract. STAT3: Signal transducer and activator of transcription 3.