Analysis Of ASF1B Expression And Prognosis In Gastric Cancer And Its Effect On Proliferation And Apoptosis Of Gastric Cancer Cells

Objective:To investigate the expression of ASF1 B in gastric cancer tissues and its relationship with prognosis,and to clarify the biological behaviour and possible mechanisms of action of ASF1 B in gastric cancer tissues.Methods: For bioinformatics analysis,we obtained m RNA data and clinical data of GC patients from the TCGA-STAD dataset and downloaded m RNA clinical data of GC patients from the GEO dataset(GSE84437,GSE29998,GSE13911)to verify the clinical value of ASF1 B.Differential expression of ASF1 B in gastric cancer tissues and normal paracancer tissues was analysed using the Wilcox rank sum test with the R software ’limma’ package,ROC curves to assess accuracy and sensitivity,the ’survival’ package and online analysis.The Kaplan-Meier survival analysis was applied to the website.The χ2 test was used for correlation analysis between ASF1 B and clinicopathological parameters in gastric cancer patients,and the Cox proportional risk model was used for univariate and multifactorial analysis of overall survival to determine the clinical value of ASF1 B in the prognosis of gastric cancer.GSEA software(Gene Set Enrichment Analysis)and WGCNA(Weighted Gene Co-expression Network Analysis)were used to screen ASF1B-related differential genes using |log2FC≧1| and P≤0.05 as cut-off criteria.Modular genes were identified by WGCNA,and genes associated with ASF1 B expression were identified by PPI co-expression network construction.Differential genes were subjected to GO(Gene Oncology)functional enrichment analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment analysis using R software,and TIMER was used to verify the correlation of ASF1 B to p53,Bax and Bcl2 online.In vitro experiments were performed using Western bolt and q RT-PCR to detect the expression of ASF1 B in gastric cancer cell lines HCG-27,MGC803,MKN45 and AGS.Two cell lines with high expression were selected for ASF1 B gene silencing and transfection efficiency was verified by Western bolt and q RT-PCR.The samples with higher transfection efficiency and non-transfected samples were selected for CCK-8 colorimetric assay,BRDU fluorescence staining and wound healing assay to assess cell proliferation ability.The effect of ASF1 B on apoptosis was examined using flow cytometry.The differences in the expression of p53,Bax and Bcl2 in the two groups were also compared by Western blot and q RT-PCR.Results: 1.Bioinformatic analysis showed that ASF1 B was highly expressed in TCGA and GEO gastric cancers relative to that in normal and adjacent tissues to the cancer(P <0.001),and the ROC curve(AUC value > 0.8)showed that ASF1 B was very sensitive and specific for predicting gastric carcinogenesis,and its expression was correlated with T stage(P < 0.05)and prognosis(P < 0.05).Cox regression analysis showed that ASF1 B could be an independent prognostic factor for gastric cancer.2.GO analysis showed enrichment in positive regulation of cell cycle,DNA integrity checkpoint,regulation of double-strand break repair and signaling pathway of p53;KEGG analysis showed enrichment in regulation of cell cycle and p53 signaling pathway.GSEA showed that ASF1 B was highly enriched in the genome of p53 signaling pathway,base excision repair,homologous recombination and mismatch repair.TIMER analysis showed that ASF1 B expression was closely associated with key genes of apoptosis: ASF1 B and TP53 were positively correlated with BAX(P <0.001)and negatively correlated with BCL2(P<0.001).3.Western blot and q RT-PCR results showed that ASF1 B was highly expressed in HCG-27 and MGC803 gastric cancer cell lines,which were applied in subsequent cell function experiments.4.CCK8 cytotoxicity assay and BRDU fluorescence staining showed that ASF1 B knockdown enhanced the proliferation of gastric cancer cells.5.Flow cytometry showed that ASF1 B knockdown reduced the apoptotic ability of gastric cancer cells,and Western blot and q RT-PCR showed that ASF1 B knockdown decreased p53/Bax and increased Bcl-2 expression.Conclusion: Gastric cancer patients with low ASF1 B expression have a poor prognosis and may be a novel biomarker to improve the prognosis of gastric cancer patients as an independent prognostic factor;knockdown of ASF1 B reduces apoptosis and promotes the proliferation of gastric cancer cells through the Bax/Bcl-2-p53 axis.