18β-glycyrrhetinic acid inhibits proliferation of gastric cancer cells through regulating the miR-345-5p/TGM2 signaling pathway.

Background: Gastric cancer (GC) is a common gastrointestinal malignancy worldwide. Based on cancer-related mortality, the current prevention and treatment strategies for GC still show poor clinical results. Therefore, it is important to find effective drug treatment targets.

Aim: To explore the molecular mechanism of 18β-glycyrrhetinic acid (18β-GRA) regulating the miR-345-5p/TGM2 signaling pathway to inhibit the proliferation of GC cells.

Methods: CCK-8 assay was used to determine the effect of 18β-GRA on the survival rate of GES-1 cells and AGS and HGC-27 cells. Cell cycle and apoptosis were detected by flow cytometry, cell migration was detected by a wound healing assay, the effect of 18β-GRA on subcutaneous tumor growth in BALB/c nude mice was investigated, and the cell autophagy level was determined by MDC staining. TMT proteomic analysis was used to detect the differentially expressed autophagy-related proteins in GC cells after 18β-GRA intervention, and then the protein-protein interaction was predicted using STRING (https://string-db.org/). MicroRNAs (miRNAs) transcriptome analysis was used to detect the miRNA differential expression profile, and use miRBase (https://www.mirbase/) and TargetScan (https://www.targetscan.org/) to predict the miRNA and complementary binding sites. Quantitative real-time polymerase chain reaction was used to detect the expression level of miRNA in 18β-GRA treated cells, and western blot was used to detect the expression of autophagy related proteins. Finally, the effect of miR-345-5p on GC cells was verified by mir-345-5p overexpression.

Results: 18β-GRA could inhibit GC cells viability, promote cell apoptosis, block cell cycle, reduce cell wound healing ability, and inhibit the GC cells growth . MDC staining results showed that 18β-GRA could promote autophagy in GC cells. By TMT proteomic analysis and miRNAs transcriptome analysis, it was concluded that 18β-GRA could down-regulate TGM2 expression and up-regulate miR-345-5p expression in GC cells. Subsequently, we verified that TGM2 is the target of miR-345-5p, and that overexpression of miR-345-5p significantly inhibited the protein expression level of TGM2. Western blot showed that the expression of autophagy-related proteins of TGM2 and p62 was significantly reduced, and LC3II, ULK1 and AMPK expression was significantly increased in GC cells treated with 18β-GRA. Overexpression of miR-345-5p not only inhibited the expression of TGM2, but also inhibited the proliferation of GC cells by promoting cell apoptosis and arresting cell cycle.

Conclusion: 18β-GRA inhibits the proliferation of GC cells and promotes autophagy by regulating the miR-345-5p/TGM2 signaling pathway.