Experimental Study On The Regulation Of Gastric Cancer Cell Proliferation,Migration And Apoptosis By ARSB Through Wnt/β-catenin Signaling Pathway

Objective:To investigate the expression level of ARSB(Arylsulfatase B)in gastric cancer(Gastric Cancer,GC)tissues and its relationship with the prognosis of gastric cancer patients,to preliminarily clarify the effect of ARSB on the value-added,migration and apoptosis of gastric cancer cells and the possible mechanism,and to provide a new theoretical basis for the early diagnosis and treatment of gastric cancer.Methods:1.The mRNA data and clinical data of gastric cancer patients were downloaded from the Gene Expression Omnibus(GEO;GSE29998,GSE54129,GSE51575)and The Cancer Genome Atlas(TCGA)databases by bioinformatics analysis.The mRNA data and clinical data from gastric cancer patients were downloaded from The Cancer Genome Atlas(TCGA)database,and the Wilcox rank sum test was used to analyze the differential expression levels of ARSB in gastric cancer patients using the "limma" package of R software;ROC curves were plotted to evaluate the precision and sensitivity.Survival" R package and Kaplan-Meier Plotter online website were used for survival analysis;the chi-square test was applied to correlate ARSB with clinical indicators of gastric cancer patients,and the Cox proportional risk model was used for univariate and multifactorial analysis of overall survival of ARSB in gastric cancer patients.clinical value in the prognosis of gastric cancer.2.In vitro cell assay,protein blotting(Western blot)was used to detect the expression of ARSB protein levels in four gastric cancer cell lines,AGS,HGC-27,MGC 803 and MKN45,and two gastric cancer cell lines with relatively high ARSB expression(AGS and HGC-27)were selected to be transfected with small interfering RNA(siRNA)to knock down The proliferation ability of gastric cancer cells after ARSB knockdown was detected by CCK-8 cell proliferation assay;the migration ability of gastric cancer cells after ARSB knockdown was assessed by cell scratch assay;the apoptosis of gastric cancer cells after ARSB knockdown was determined by flow cytometry;and the Western blot was used to verify that ARSB The expression of genes downstream of WNT pathway was verified by Western blot.3.Experiments on the effect of Wnt/β-catenin pathway inhibitor XAV939 on the biological characteristics and behavior of gastric cancer cells.two types of gastric cancer cells,AGS and HGC-27,were each divided into four groups:(i)blank control group: XAV939(-)and ARSB siRNA(-);(ii)interference alone group: ARSB siRNA(+)and XAV939(-);(iii)inhibitor alone group: XAV939(+)and ARSB siRNA(-);(iv)interference combined with inhibitor group: ARSB siRNA(+)and XAV939(+).The effect of XAV939,an inhibitor of Wnt/β-catenin signaling pathway,on ARSB protein expression after treatment of gastric cancer cells was examined by Western blot;to investigate whether direct interference with Wnt signaling pathway would affect the function of gastric cancer cell lines;to examine the value-added and migration of gastric cancer cells in each group using CCK-8 cell proliferation assay and cell scoring assay,respectively.Results:1.Analysis of GEO and TCGA databases showed that ARSB was highly expressed in unpaired gastric cancer tissues compared with normal tissues(P < 0.05);similarly,ARSB was highly expressed in paired tissues compared with normal tissues next to cancer(P < 0.001);ROC curves showed that ARSB was highly accurate in predicting gastric carcinogenesis;survival The results of survival analysis showed that the overall survival,progression-free survival and progression survival of patients with high ARSB expression were significantly shorter compared with those with low expression;clinicopathological correlation analysis suggested that ARSB correlated with the clinical index T-stage of gastric cancer patients;Cox regression analysis showed that ARSB could be an independent risk factor affecting the prognosis of gastric cancer patients.2.Western blot results indicated that ARSB was significantly expressed in AGS and HGC-27 cells,and it was selected for subsequent cell function experiments.The results of CCK-8 cell proliferation experiment showed that knockdown of ARSB significantly inhibited the proliferation of gastric cancer cells.The cell scratch test showed that the migration ability of gastric cancer cells was significantly weakened after ARSB knockdown.Flow cytometry showed that knockdown of ARSB could promote apoptosis of gastric cancer cells.Western blot analysis showed that knockdown of ARSB inhibited the expression ofβ-catenin and c-Myc related genes in WNT signaling pathway.3.Western blot results suggested that the expression level of ARSB protein was significantly downregulated in both gastric cancer cells after treatment with Wnt/β-catenin pathway inhibitor XAV939;knockdown of ARSB combined with Wnt/β-catenin pathway inhibitor XAV939 further inhibited the expression of β-catenin and c-Myc;CCK-8 assay The results of CCK-8 assay showed that the proliferation ability of cells in both the interference alone and the inhibitor alone groups was reduced compared to the blank control group(P < 0.01),and the proliferation ability of cells in the interference combined with the inhibitor group was more significantly reduced compared to the blank control group(P < 0.001);the results of cell scratch assay suggested that the migration ability of cells in the interference alone or the inhibitor alone group was reduced compared to the blank control group(P < 0.01),and the migration ability of cells in the interference combined with the inhibitor group was reduced compared to the blank control group(P< 0.01).The migration ability of the cells in the interfering group and the inhibitor group was more significantly reduced compared with the blank control group(P < 0.001).Conclusion:The survival time of gastric cancer patients with significantly high expression of ARSB was shorter.This gene is an independent prognostic risk factor for gastric cancer.ARSB knockdown could inhibit the proliferation and migration of AGS and HGC-27 cells and promote the apoptosis of gastric cancer cells.ARSB promotes malignant biological behavior of gastric cancer cells by regulating Wnt/β-catenin signaling pathway.These results suggest that ARSB plays an important role in the occurrence and development of gastric cancer.