The Role Of GKN2 On The Biological Functions Of Gastric Cancer And Mechanism Analysis

Objective:It's aim to discuss the role of GKN2 on proliferation,cell cycle,migration and invasion in gastric cancer cells,and have preliminary illustration on the inhibitory effect of GKN2 on which may provide theoretical basis for the linical design of effective strategy of gastric cancer.Method:RT-PCR were used to detect the m RNA expression of GKN2 in 8 gastric cancer tissues together with paired normal tissues and 6 gastric cancer cells together with normal gastric cell,and the m RNA expression level of GKN1,TFF1 and TFF2 in the GKN2 overexpression cells and control cells;Western Blot were used to detect the protein expression of GKN2 in 8 gastric cancer tissues together with paired normal tissues and 6 gastric cancer cells together with normal gastric cell,and the protein level of GKN1,the major genes of the biological function and signaling pathway;GKN2 overexpression cell models was built up by lentiviral vector system.Then,RTCA,clone forming experiments and wound-healing assay,migration/invasion assay respectively were used to study the role of GKN2 in proliferation and invasion of gastric cancer in vitro.The gene expression profiling assay was performed to identify the differences in gene expression profiles between GKN2 overexpression group(AGS_GKN2)and control group(AGS_con).GO analysis and KEGG analysis were used to calculate biological pathways about the biological function of gastric cancer.Results:1.RT-PCR and Western Blot show that GKN2 expression level of gastric cancer tissue and gastric cancer cells is much lower than paired normal tissues and gastric epithelium cell.2.RTCA and clone formation experienment show that overexpression of GKN2 can reduce cell proliferation ability by RTCA in vitro.Western Blot analyses were performed to detect the effects of GKN2 inhibiting on the expression of proliferation-related protein including PCNA,Survivin and BCL-2.Flow cytometry show that overexpression of GKN2 can reduce the percentage of S phase cells in AGS and arrest the G0/G1 phase in SGC-7901.Western Blot shows that GKN2 can reduce the expression level of cyclin A and increase P21 in AGS and reduce the expression level of cyclin E,cyclin A and cyclin D and increase P21 in SGC-7901.3.Wound-healing assay,migration/invasion assay show that overexpression of GKN2 can depressed cell migration and invasion in vitro.Western blot analyses were performed to detect the effects of GKN2 inhibiting on the expression of metastasis-related proteins including MMP7,MMP9,MMP2 and uprelating on the expression of Timp2.4.The results of gene expression profing assays(Only testing AGS gastric cancer cells)showed that 32 different genes were changed in AGS_GKN2correspond to AGS_con(foldchange ? 3 or ? 0.33).13 genes were up-regulated(foldchange ? 3)and 19 genes were down-regulated(foldchange ?0.33).There were 55 high enrichment factor pathways by KEGG analysis and PI3K/AKT/PTEN/m TOR pathways belongs to them.Conclusion:1.The GKN2 expression level of gastric cancer tissue and gastric cancer cells(MKN45,MKN28,BGC-823,NCI-N87,SGC-7901,AGS)is much lower than paired normal tissues and gastric epithelium cell relatively;2.GKN2 dramatically inhibit the proliferation of gastric cancer cell by lowering the expression of PCNA,Survivin and Bcl-2 and downregulate the expression of cycle protein,upregulate cycle protein kinase inhibition P21,GKN2 significantly inhibit the metastasis ability by reduce the abundance of MMP2,MMP7,MMP9 and up-regulate TIMP2 in the gastric cancer cell.3.GKN2 inhibit gastric cancer cell malignant behavior by downregulating the activation level of PI3K/AKT/PTEN/m TOR and JAK/STAT signaling pathways.4.GKN2 is a potential tumor suppressor gene and expected to become the molecular targets for the prevention and treatment of gastric cancer.