| Objectives: To investigate the effects of GTPBP4 expression on the biological behaviors of gastric cancer cells in vitro and the possible mechanism of GTPBP4 in the pathogenesis of gastric cancer.Methods 1.Differentially expressed genes of gastric cancer tissues and cancer adjacent tissues were detected by gene chip in 30 patients with gastric cancer.Overexpression of GTPBP4 gene in gastric cancer tissues was identified by high content screening(HCS).The correlation between GTPBP4 overexpression and tumor stage and prognosis was assessed through analysis clinical pathological data.2.The expression of GTPBP4 in gastric cancer cell lines and mucosal cells was detected by real-time quantitative PCR.To determine the potential role of GTPBP4 gene in gastric cancer,the lentiviral vector carrying GTPBP4-specific sh RNA was transfected into AGS or MKN-45 gastric cancer cells;and the interference efficiency was detected by real-time quantitative PCR.The effects of GTPBP4 gene expression on the proliferation,apoptosis and cloning of AGS and MKN-45 cells were evaluated by MTT,flow cytometry and cell cloning assays,respectively.3.To investigate which signal pathway was involved in the roles of GTPBP4 gene in gastric cancer cells,gene chip assay was conducted in MKN-45 cells after down-regulation of GTPBP4 expression.The expression of candidate genes was then confirmed by Western blot.Results 1.A total of 1745 genes were differentially expression after gene chip screening,among which 889 genes were up-regulated and 856 were down-regulated.20 high-expression genes were selected for HCS screening and the results showed that four genes were positive for proliferation(Fold change ≥1.8).After searching NCBI literature database,clinical tumor staging and prognosis,the candidate gene GTPBP4 was finally identified for further investigation.The expression of GTPBP4 was determined in 65%(26/40)of gastric cancer tissues and 32.5%(13/40)in adjacent tissues,respectively(P <0.05).The expression of GTPBP4 was significantly correlated with tumor depth,lymph node metastasis and tumor stage(P <0.05),but not with age,sex and differentiation in gastric cancer cases.2.The expression of GTPBP4 gene was highly expressed in gastric cancer cell lines,and significantly higher than that in normal gastric mucosal cells(P <0.05).After transfection with sh RNA-lentivirus,the m RNA expression of GTPBP4 gene in MKN-45 cells and AGS cells was inhibited(P <0.05),and with the interference efficiency of 80.7% and 85.3% respectively.After down-regulation of GTPBP4 expression,cell proliferation and the clone formation were decreased while cell apoptosis was increased in both AGS and MKN-45 cells(P <0.05).3.The Gene chip results showed that there were 1144 differentially expressed genes in MKN-45 cells after down-regulation of GTPBP4,among which 757 were up-regulated and 387 genes were down-regulated.Enrichment analysis showed that there were several genes involving in P53 signaling pathway,such as PIK3R3,HDAC9,TP53INP1,KAT2 B,THBS1,IRS1,TNFRSF10 B,RRM2B,PERP,MDM2,HIF1 A,TRIM29,CCND1,FAS,SERPINE2,PTEN and so on.The expression of related proteins in P53 signaling pathway was further detected by Western blot and found CCND1 expression was down-regulated while P53,TNFRSF10 B,MDM2,FAS,PTEN and CDKN1 A were up-regulated after down-regulation of GTPBP4 in gastric cancer cells.Through bioinformatics analysis,we speculated that GTPBP4 may play a role by regulating P53 signal pathway.Conclusions 1.GTPBP4 is highly expressed in gastric cancer tissues and cell lines.High expression of GTPBP4 is closely related to gastric cancer staging,invasion and lymph node metastasis.2.After down-regulation of GTPBP4,cell proliferation and clone formation are decreased and cell apoptosis is increased in MKN-45 cells and AGS cells.3.GTPBP4 may involve in the initiation and progression of gastric cancer through regulating P53 signaling pathway. |
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