SPAG6 Silencing Regulates The Apoptosis Of SKM-1 Cell And Its Mechanism

Objective:To construct the pGC-shRNA-SPAG6(short hairpin RNA)lentivirus targeting silence SPAG6 gene,and explore the relationship between SPAG6 and PTEN,and investigate whether SPAG6 induce the apoptosis of myelodysplastic syndrome(MDS)cell line SKM-1 in vitro and in vivo.Methods:After lentivirus-mediated knockdown of SPAG6 in SKM-1 cell,then fluorescence was observed under a fluorescence microscope.The transfection efficiencies were measured by flow cytometry,and qRT-PCR?western blot were detected the inhibitory degree of SPAG6.Meanwhile,explore the influence between the expression of SPAG6 and activation of PI3K/AKT signal pathway.Then the SKM-1 cell with SPAG6 shRNA lentivirus were treated with the PI3K inhibitor Ly294002 or pan caspase inhibitor z-VAD-fink and the apoptosis rates were measured by FACS,and the expressions of related proteins were examined by western blot analysis.Xenograft models were successfully established.The tumor volume was calculated and the cell apoptosis was detected using TUNEL assays.Western blot and IHC were used to observed the activation of PI3K/AKT signal pathway in vivoResult:SPAG6-shRNA lentiviral vector were successfully constructed.The lentivirus-mediated knockdown of SPAG6 in SKM-1 cells increased PTEN expression and AKT inactivation,which in turn resulted in cell apoptosis as evidenced by Mcl-1 downregulation,cytochrome c release,and active caspase-9 expression.Consistently,the PI3K inhibitor Ly294002 synergistically enhanced apoptosis of SKM-1 cells when co-administered with SPAG6 shRNA lentivirus.Moreover,treatment with the pan caspase inhibitor z-VAD-fmk failed to prevent PTEN activation upon SPAG6 knockdown,suggesting that SPAG6-regulated PTEN expression was caspase activation-independent.Furthermore,tumor tissues from NOD/SCID mice inoculated with SPAG6-shRNA lentivirus pre-infected SKM-1 cells exhibited significantly elevated apoptosis in both the extrinsic and intrinsic pathways.Conclusion:SPAG6 silencing induces PTEN expression to regulate apoptosis though the PI3K/AKT pathway,indicating that SPAG6 could be a potential therapeutic target for MDS.