The Relationship Between SPAG6 And Cell Apoptosis In Myelodysplastic Syndrome And Its Possible Mechanism

Objective To explore the relationship and possible mechanism between SPAG6 and cell apoptosis in MDS through the pGC-shRNA-SPAG6(short hairpin RNA)lentivirus.Methods1.Verification the interference efficiency of SPAG6-shRNA lentivirus against human SPAG6 gene.The SKM-1 cells were infected with the SPAG6-shRNA lentivirus or NC-shRNA lentivirus respectively,and then fluorescence was observed under a fluorescence microscope.The transfection efficiencies were measured by flow cytometry,and the inhibitory degree of SPAG6 was measured by qRT-PCR and western blot analysis.2.Explore the influence between the expression of SPAG6 and activation of the TRAIL signal pathway.After lentivirus were transfected into SKM-1 cells,the apoptosis rates were measured by FACS,and the expressions of related proteins were examined by western blot analysis.3.Investigate the dependence on the expression level of SPAG6 in TRAIL signal pathway.SKM-1 cells were treated with different concentrations of recombinant human sTRAIL(r TRAIL),while SKM-1,K562 and U937 cells were treated with the same concentration of rTRAIL,respectively.The apoptosis rates were measured by FACS,and cell proliferation was examined by CCK-8 assay.The expressions of related proteins were examined by western blot analysis.4.Explore the mechanism and the key molecules in TRAIL signal pathway regulated by SPAG6.The expressions TRAIL death receptors were detected by qRT-PCR and western blot analysis.And immunoprecipitation was used to explore the relationship between the FADD and TRAIL death receptors.Results1.Lentivirus-mediated SPAG6 silence in SKM-1 cells.An abundance of cells with green fluorescence could be observed under a fluorescence microscope.The transfection efficiencies were 97.19%±3.21% and 88.31%±12.81%,respectively.And both the mRNA and protein levels were reduced in the SKM-1 cells infected with SPAG6 shRNA-lentivirus as compared to the control group.2.Reducing the expression of SPAG6 activates the TRAIL signal pathway.The apoptosis rates in SKM-1 infected with SPAG6-shRNA were significantly higher than those in the control group(NC-shRNA 8.65%±2.07% vs.SPAG6-shRNA 20.20% ±2.11%,P<0.05).Western blot analysis revealed that the expression levels of cleaved PARP,caspase-8,and cleaved caspase-8 were obviously higher in SKM-1 infected with SPAG6-shRNA lentivirus than in the control group,whereas the expression of PARP showed no significant difference between the two groups.In addition,the expression of apoptotic factors,including BAK,BAX,BID and caspase-3,also showed statistical differences in the two groups.3.SPAG6 resists apoptosis induced by TRAIL.Following r TRAIL treatment,the flow cytometry data indicated that the higher concentration of TRAIL led to higher apoptosis rates.And cell proliferation was suppressed in the cells treated with TRAIL as compared to the normal control group.Furthermore,the western blot assay showed that the expressions of related proteins were higher after treatment.However,in the SKM-1,U937 and K562 cells after treatment with the same concentration of r TRAIL,the apoptosis rates were not significantly different in U937 and K562,and the same results were also shown in the CCK-8 assays.4.SPAG6 can influence the interaction between FADD and TRAIL death receptors.After infection,both the mRNA and protein levels of the two receptors were no changes,while the expression of FADD was statistically different.And the result of immunoprecipitation showed that the interaction between FADD and death receptors increased in SKM-1 infected with SPAG6 shRNA-lentivirus as compared to the control group.ConclusionThis study demonstrates that SPAG6 may regulate apoptosis through the TRAIL signal pathway by inhibiting the expression of FADD and the interactions between FADD and the TRAIL death receptors.