The Role And Mechanisms Of Suppression Of SHIP2 Mediated By Downregulation Of Sp1 In Human Gastric Cancer Cells

Background & Objective: SHIP2(src-homology 2-containing inositol 5 –phosphatase 2)is a member of inositol polyphosphate 5-phosphatase family.It has been implicated in human type ? diabetes,arthrosclerosis,and cancer.However,the role of SHIP2 in human gastric cancer remains unclear so far.In our previous study,it has been shown that SHIP2 was commonly downregulated in gastric cancer compared with normal gastric mucosa,and reduced SHIP2 expression promotes tumorigenesis and proliferation of gastric cancer via activation of the PI3K/Akt signaling.Based on these,the aim of our study is to explore the molecular mechanisms of the downregulation of SHIP2 in gastric cancer cells.Methods: The gene copy number variation of SHIP2 in normal gastric epithelial cells and a pan of gastric cancer cells were detected by real-time quantitative PCR.The exons mutations of SHIP2 were analyzed by exon sequencing.The expression levels of SHIP2 in normal gastric epithelial cells and gastric cancer cells treated with 10 ?M DNA methylation inhibitor 5–aza-d C and 5 ?M histone deacetylase inhibitor SAHA were detected by western blot,respectively.The promter methylation levels of SHIP2 were detected by BSP(bisulfite sequencing PCR)assays.The transcriptional activity of SHIP2 promoter was determined by dual luciferase reporter gene system.The predicted transcription factor binding sites of the minimum fragment with transcription activity of SHIP2 promter were determined by bioinformatics analysis.Furthermore,the role of the predicted transcription factor Sp1 was clarified by use of the deletion of these sites and dual luciferase reporter gene assay system.The expression levels of Sp1 and SHIP2 in gastric cancer cells and normal gastric epithelial cell were detected by q RT-PCR,western blot and immunohistochemical,respectively.SGC-7901 and MGC-803 cells with the lowest expression of Sp1 were transiently transfected with Sp1 c DNA.The cell growth and proliferation of gastric cancer cells were detected by colony formation assays ad MTS assays.The apoptotic cells were detected by FCM stainning with PI.The cell migration and invasion of gastric cancer cells were analyzed by the wound healing assays and tranwell assays,respectively.The combination of Sp1 and SHIP2 was determined by Ch IP assays.The m RNA expression levels of SHIP2 in gastric cancer cells transfected with Sp1 c DNA were detected by q RT-PCR.Results: 1)There was no statistical difference in the gene copy number variation of SHIP2 in gastric cancer cells compared with normal gastric epithelial cells.There are different levels of the base mutations and deletions of SHIP2 exons in gastric cancer cells except for HGC-27 cells.Although 5-aza-d C but not SAHA upregulated SHIP2 expression in gastric cancer cells,the methylation frequency of SHIP2 promoter was very low.The minimum transcription activity region of SHIP2 promoter was from-111 to-63 bp,and there were two Sp1-binding sites in this region.The transcription activity of SHIP2 promoter decreased significantly upon deletion of these two Sp1-binding sites.2)The expression of Sp1 and SHIP2 in gastric cancer cells were commonly downregulated in comparison of normal gastric epithelial cells.3)Overexpression of SHIP2 inhibited cell proliferation,induced apoptosis,suppressed cell motility and invasion in gastric cancer cells.4)The transcriptional activities of SHIP2 were increased significantly in gastric cancer cells overexpressing Sp1.Conclusion: The suppression of SHIP2 mediated by downregulation of Sp1 is one of the important molecular mechanisms of malignant biological behavior in human gastric cancer cells.