Mechanisms By Which WYC-209 Down-regulates WNT4 Gene To Suppress Malignant Phenotype Of Gastric Cancer Cells Through Wnt/β-catenin Pathway

Objective This study aims to investigate the mechanism by which WYC-209 inhibits gastric cancer progression in the Wnt/β-catenin signaling pathway,to provide a theoretical basis for the role of WYC-209 in inhibiting gastric cancer,and to provide new ideas for the clinical application of retinoic acid analogues.Methods In this study,MTT,Transwell and clone formation assays were used to investigate the effects of the drug on the toxicity of gastric cancer cells,on the malignant phenotype such as migration invasion and proliferation ability of gastric cancer cells.Then,qRT-PCR and Western Blotting assays were used to investigate the effects of WYC-209 on epithelial mesenchymal transition and expression levels of proteins related to signaling pathways in gastric cancer cells.In this study,we used second-generation high-throughput sequencing to analyze the differential mRNA expression of genes in HGC-27 cells treated with WYC-209,and further analyzed the differential genes and pathways.Total proteins from HGC-27 cells treated with WYC209 were extracted,and qRT-PCR was used to verify the selection of the most likely related gene protein,WNT4 gene,based on nine differential genes selected from the literature and databases,and subsequent Western Blotting assays were performed using this gene protein to verify whether expression changes were observed.Using the HGC-27 gastric cancer cell line as a model,the WNT4 overexpression plasmid and subsequent Transwell,MTT and clone formation assays were used to investigate the role of WNT4 in the reversion of the malignant phenotype of gastric cancer cells,and thus to test the hypothesis that WNT4 plays a role in the inhibition of the malignant biological behavior of gastric cancer cells by WYC-209.Results The proliferation ability and invasive metastasis of gastric cancer cells were significantly reduced after WYC-209 treatment.Compared with the control group,HGC-27 and AGS cells in WYC-209-treated group suggested up-regulation of Ecadherin expression and down-regulation of N-cadherin,Vimentin and Snail expression in the results of real-time PCR and Western Blotting experiments.In the follow-up experiments,the sequencing trend of WNT4 was found to be consistent with the PCR validation trend by second-generation high-throughput RNA sequencing and qRT-PCR validation,and the results were validated by Western Blotting in the follow-up experiments,which indicated that WYC-209 could inhibit the expression of WNT4 protein and the activation of Wnt/β-catenin signaling pathway by inhibiting the expression of WNT4 protein.The results indicated that WYC-209 could suppress the malignant phenotype of gastric cancer cells by inhibiting WNT4 protein expression and Wnt/β-catenin signaling pathway activation.In the cell function and mechanism rescue experiments using WNT4 transfected plasmid in combination with WYC-209 and WYC-209 alone,it was shown that overexpression of WNT4 was able to restore the inhibitory effect of WYC-209 treatment on cell proliferation and migration-invasive ability.Conclusion Taken together,the experiments showed that WYC-209 was able to suppress the malignant phenotype of gastric cancer cells and delay the progression of gastric cancer by inhibiting the expression of WNT4 in the Wnt/β-catenin signaling pathway.This study demonstrated that the theoretical basis of the inhibitory effect of WYC-209 on gastric cancer was further refined,providing evidence for the clinical application of WYC-209 in gastric cancer.