| ObjectiveThis study explored the effects of ZIC2 expression in osteosarcoma,proliferation and invasion of osteosarcoma cells and related mechanisms,aiming to lay a theoretical and experimental basis for the search for new molecular targets of osteosarcoma.Methods(1)Detection of ZIC2 expression in osteosarcoma tissues:Osteosarcoma tissues and adjacent normal tissues were collected,and ZIC2 mRNA expression in the above tissue samples was examined.Detection of ZIC2 expression in osteosarcoma cell lines:detection of ZIC2 mRNA and protein expression in human osteosarcoma U20S,Saos2,MG63 cell lines and human osteoblast cell line hFOB1.19.Effects of ZIC2 on proliferation and invasion of osteosarcoma cells and expression of related proteins:The expression of ZIC2 in osteosarcoma MG63 cells was up-regulated or down-regulated by prion vector transfection,and the effect of ZIC2 on proliferation and invasion of MG63 cells was detected.The effect of ZIC2 expression on the expression of E-cadherin,Twistl,Snaill,Snail2,MMP2,MMP9,Cyclin D1 protein in MG63 cells.(2)Effect of ZIC2 on the expression of SHIP2 in osteosarcoma cells:The expression of ZIC2 in MG63 cells was up-regulated or down-regulated by transfection of lentiviral vector,and the expression of SHIP2 mRNA and protein and ZIC2 protein in ZIC2 expression was detected.Effect of SHIP2 on the expression of PI3K/Akt signaling pathway-associated proteins in osteosarcoma cells:The expression of p-PI3acK,PI3K,p-Akt,Akt and ZIC2 in MG63 cells was detected by up-regulation and down-regulation of ZIC2 expression.The lentiviral vector carrying the ZIC2 overexpression gene or the ZIC2 overexpression gene + SHIP2 overexpression gene was transfected into 293T cells,and the virus solution was collected after the cells were packaged with lentivirus,and then the virus solution was infected into MG63 cells,and each group of cells was detected.Expression of p-PI3acK,PI3K,p-Akt,Akt,ZIC2 proteins.The lentiviral vector carrying the ZIC2 overexpression gene was transfected into 293T cells,and the virus solution was collected after the cells were packaged with lentivirus,and then the virus solution was infected with MG63 cells.The cells were randomly divided into 2 groups,one of which was added to the PI3K/Akt signaling pathway inhibitor(LY294002).The expressions of E-cadherin,Twistl,Snaill,Snail2,MMP2,MMP9,Cyclin D1 and ZIC2 proteins in each group were detected,and the proliferation activity of each group was detected.Results(1)Detection of ZIC2 expression in osteosarcoma:The expression level of ZIC2 mRNA in osteosarcoma was significantly higher than that in adjacent normal tissues(P<0.001).Detection of ZIC2 expression in osteosarcoma cell lines:The expression level of ZIC2 mRNA in three osteosarcoma cells was significantly higher than that of hFOB1.19 cells.At the same time,the expression of ZIC2 protein was similarly detected by western blot.The expression of ZIC2 protein in human osteosarcoma U20S,Saos2,MG63 and T24 cell lines was significantly higher than that of human osteoblast cell line hFOB1.19.The effect of ZIC2 on the proliferation and invasion of osteosarcoma cells and the expression of related proteins:After the up-regulation of ZIC2,the proliferation activity of MG63 cells increased significantly(P<0.05),and the number of clones and the number of invasive cells increased significantly(P<0.001).After ZIC2 expression was down-regulated,the proliferation activity of MG63 cells was significantly decreased(P<0.05),and the number of clones and the number of invasive cells were significantly decreased(P<0.001).After ZIC2 expression was up-regulated,the expression level of E-cadherin protein in MG63 cells was significantly decreased,while the expression levels of Twistl,Snail1,Snail2,MMP2,MMP9 and Cyclin D1 protein were significantly increased.After ZIC2 expression was down-regulated,E-cadherin in MG63 cells was down-regulated.The expression level of the protein was significantly increased,while the expression levels of Twistl,Snail 1,Snail2,MMP2,MMP9,and Cyclin D1 protein were significantly decreased(2)Effect of ZIC2 on the expression of SHIP2 in osteosarcoma cells:After up-regulation of ZIC2 expression,the expression levels of SHIP2 mRNA and protein in MG63 cells were significantly decreased(P<0.001).After down-regulation of ZIC2 expression,the expression of SHIP2 mRNA and protein in MG63 cells was down-regulated.The level was significantly increased(P<0.01).The effect of SHIP2 on the expression of PI3K/Akt signaling pathway-associated proteins in osteosarcoma cells:Compared with the control group,the expression of PI3K and Akt in the siZIC2 and oeZIC2 cells did not change significantly,while the siZIC2 group p-PI3K/PI3K and p-The ratio of Akt/Akt was significantly lower than that of the control group.The ratio of p-PI3K/PI3K and p-Akt/Akt in the oeZIC2 group was significantly higher than that in the siZLC2 group.Compared with the control group,the expression of PI3K and Akt in the oeZIC2 group and the oeZIC2+oeSHI02 group did not change significantly.The ratio of p-PI3K/PI3K and p-Akt/Akt in the oeZIC2 group was significantly higher than that in the control group,while oeZIC2+oeSHI02 The ratio of p-PI3K/PI3K to p-Akt/Akt was significantly reduced in the cells.Compared with the control group,the expression level of E-cadherin protein in the oeZIC2 group was significantly decreased,while the expression level of E-cadherin protein in the oeZIC2+LY294002 group was significantly higher;compared with the control group,Twistl in the oeZIC2 group.The expression levels of Snaill,Snail2,MMP2,MMP9 and Cyclin D1 protein were significantly increased,while the expression levels of Twistl,Snaill,Snail2,MMP2,MMP9 and Cyclin D1 proteins in oeZIC2+LY294002 group were significantly decreased.Compared with the control group,the proliferation activity of cells in the oeZIC2 group was significantly increased on the 2nd,3rd,and 4th day,while the proliferation activity of the cells in the oeZIC2+LY294002 group was significantly decreased on the 2nd,3rd,and 4th day.ConclusionZIC2 is highly expressed in human osteosarcoma cells and tissues,and its overexpression promotes proliferation and invasion of osteosarcoma cells by down-regulating SHIP2 and activating PI3K/AKT signaling pathway. |
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