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

Abstract

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 in vivo. 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.

Keywords: 18β-glycyrrhetinic acid; Autophagy; Gastric cancer; MiR-345-5p; Proliferation; TGM2.