To Explore The Mechanism Of 18β-glycyrrhetinic Acid Inhibiting Proliferation Of Gastric Cancer Cells Based On MiR-204-3p/MAPK Signaling Pathway

Objective To explore the molecular mechanism of 18β-glycyrrhetinic acid inhibiting the proliferation of gastric cancer cellsMethods Firstly,the potential targets and molecular mechanisms of glycyrrhetinic acid(GRA)in the treatment of gastric cancer were predicted by bioinformatics.cell phenotype assay,q RT-PCR and Western blot were used to detect the changes of related cell phenotype,genes and proteins in gastric cancer cells after 18β-GRA intervention,an immunofluorescence chemical assay was used to detect the nuclear transcription of related proteins in gastric cancer cells after 18β-GRA intervention,and subcutaneous tumor formation in BALB/c nude mice to verify the effect of 18β-GRA on gastric cancer cells.Then,mi RNAs differentially expressed in gastric cancer cells after 18β-GRA intervention were screened by mi RNAs transcriptomic analysis,and their expression levels were detected by q RT-PCR.Lentivirus transfection was used to up-regulate mi R-204-3p in gastric cancer cells.The effect of upregulateing mi R-204-3p on cell proliferation was detected by CCK-8,clonay formation assay was used to detect cell colony formation ability,the effect of cell cycle and apoptosis were detected by flow cytometry,and a subcutaneous tumorigenic model of BALB/c nude mice was established to verify the effect of up-regulateing mi R-204-3p on tumorigenic ability of gastric cancer cells.Based on the previous research group,we have confirmed the targeting relationship between mi R-204-3p and KRAS,q RT-PCR and Western blot were used to detect the influence of overexpression of mi R-204-3p on related targets of MAPK signaling pathway.The effects of up-regulateing mi R-204-3p on apoptosis-related proteins and necroptosisrelated proteins were detected by Western blot.Results The results of bioinformatics analysis showed that 156 common targets were obtained of GRA treat gastric cancer,among which TNF,IL-6,CTNNB1,PPARG,PTGS2,ESR1,MAPK3,PPARA,AR and CYP19A1 were relatively important core targets.GEPIA database was used to analyze the core targets expression in gastric cancer and normal tissues,and it was showed that CTNNB1 was significantly up-regulated in gastric cancer tissues.Meanwhile,the role of core targets in the staging of gastric cancer was analyzed,and it was found that TNF,PPARG and ESR1 played an important role in the tumor staging of patients with gastric cancer.Kaplan-Meier plotter database analysis showed that TNF,ESR1,MAPK3,PPARA,AR,CTNNB1 and PPARG could be used as prognostic markers for gastric cancer.Then,18α-GRA,18β-GRA and the core targets were assessed to have good binding activity by molecular docking.The results of bioinformatics were analyzed comprehensively,and the effect of 18β-GRA on the MAPK signaling pathway was verified by cell experiments.The results showed that 18β-GRA could inhibit the proliferation of gastric cancer cells,promote cell apoptosis,block cell cycle,and inhibit the ability of cell colony formation(p < 0.001).18β-GRA can inhibit the expression of KRAS and p-ERK1/2 in the MAPK signaling pathway(p < 0.01),and inhibit the nuclear transcription of p-ERK1/2(p < 0.01).In addition,18β-GRA inhibited the effect of subcutaneous tumor formation and inhibited the expression of KRAS and p-ERK1/2 in tumor tissue in BALB/c nude mice(p < 0.01).mi R-204-3p expression was significantly up-regulated after 18β-GRA intervention in gastric cancer cells(p = 0.0017234),and the results of q RT-PCR were consistent with the results of mi RNAs transcriptomic analysis(p < 0.05).The expression level of mi R-204-3p in AGS,HGC-27 and MKN-45 cells was lower than normal gastric mucosal epithelial GES-1cells(p < 0.001).Lentivirus transfected gastric cancer cells to overexpressed mi R-204-3p in gastric cancer cells.The results of CCK-8 cell proliferation experiment showed that overexpression of mi R-204-3p could inhibit the proliferation of gastric cancer cells(p < 0.05),the results of cloning formation experiment showed that overexpression of mi R-204-3p could inhibit the colony formation ability of gastric cancer cells(p < 0.001),flow cytometry showed that overexpression of mi R-204-3p could arrest the cell cycle in G0/G1 phase(p < 0.01)and promote cell apoptosis(p < 0.001),and the results of subcutaneous tumor formation in BALB/c nude mice showed that overexpression of mi R-204-3p inhibited the growth of subcutaneous tumor in BALB/c nude mice(p <0.001).Lentivirus transfected gastric cancer cells,and q RT-PCR revealed that overexpression of mi R-204-3p significantly down-regulated KRAS,ERK1 and ERK2 in gastric cancer cells(p <0.05).Western blot showed that overexpression of mi R-204-3p inhibited the KRAS and downstream effector p-ERK1/2 in the MAPK signaling pathway(p < 0.01).Lentivirus transfected gastric cancer cells to overexpressed mi R-204-3p,and Western blot detected the apoptosis-related proteins and necroptosis-related proteins.In terms of apoptosis,the results showed that overexpression of mi R-204-3p decreased the expression of BCL-2(p < 0.01),while BAX protein was not change(p > 0.05),Caspase-3 protein and BCL-2/BAX was decreased(p < 0.01).In terms of necroptosis,overexpression of mi R-204-3p decreased the expressions of P-RIP1 and P-MLKL(p < 0.01),while the expressions of RIP1 and MLKL were not changed(p > 0.05).Conclusion1.18β-GRA can inhibit the activity of gastric cancer cells,promote cell apoptosis,block cell cycle,inhibit cell colony formation and the growth of subcutaneous tumor.2.18β-GRA inhibits proliferation of gastric cancer cells through MAPK signaling pathway3.18β-GRA up-regulates mi R-204-3p and inhibits MAPK signaling pathway to inhibit the proliferation of gastric cancer cells.4.mi R-204-3p promotes apoptosis of gastric cancer cells by inhibiting MAPK signaling pathway and necrotizing apoptosis pathway,thus inhibiting proliferation of gastric cancer cells.