Effect Of HSHIP On Proliferation And Apoptosis Of Hela Cells Through Downregulation Of PI3K/Akt Pathway

Background:Human SH2-containing inositol-5-phosphatase(hSHIP)belongs to inositol phosphatase family and restricts to hematopoietic cells.The studies showed that hSHIP acts as a negative regulator of proliferation,survival and end cell activation in hematopoietic cells.To explore whether hSHIP induces apoptosis and cell cycle arrest in nonhaemopoietic tumors with abnormal activation of PI3K/Akt.Methods:The full-length SHIP cDNA fragment was amplified by RT-PCR from the human peripheral blood mononuclear cells and subcloned into pcDNA3.1-GFP vector to construct pcDNA3.1-hSHIP-GFP full-length vector.pcDNA3.1-hSHIP-GFP and pcDNA3.1-GFP(control)were transfected into HeLa cells by Lipofectamine^TM2000,The stable transfected HeLa cell lines were then established by screening culture with G418,and the expression of hSHIP-GFP and GFP were identified by Flurescent microscope and Western blotting.Cell cycle was examined by Flow cytometry.Apoptosis was detected by Hoechst33258/ PI and caspase3 activity assay.Cell growth was determined by MTT assay and tumorigenicity in nude mice.The levels of Akt expression were measured by semi-quantitative RT-PCR and Western blotting,and phosphorylation of Akt(Ser473)was abserved by ELISA.Results:The eukaryotic expression vector pcDNA3.1-hSHIP-GFP were contracted successfully,and stable transfected HeLa cell lines were established.Flow cytometry analysis showed that expression of hSHIP remarkably increased the proportion of HeLa cells in S-phase(from 27.3±4.50%to 47.2±7.78%,P＜0.05)and decreased the proportion of cells in G₂/M phase(from 14.5±1.91%to 2.6±0.71%, P＜0.05),these data showed that hSHIP significantly induced S-phase arrest of HeLa cells.hSHIP-induced apoptosis reflected by Hoechst33258/PI and caspase-3 activity assay.The cell growth was reduced by 80.19%(P＜0.05)at 96h,and the tumorigenicity in nude mice was reduced evidently(255.0±50mg vs 117.5±35mg, P＜0.05),the ratio of inhibition of tumor was 53.92%(P＜0.05),Further tests showed that hSHIP leads to down-regulate expression of Akt and phosphorylation of Akt.Conclusion:In this study,hSHIP can significantly inhibit proliferation and survival of HeLa cells,and induce S arrest and apoptosis.The possible mechanism is related to downregulation of the expression and phosphorylation of Akt.These results would contribute to the further study of its anti-tumor biological functions and the suppression of other nonhaemopoietic tumors with abnormal activation of PI3K/Akt by hSHIP.