| BackgroundAdult T-cell leukemia(ATL)is a malignant T lymphocytic proliferative disease that is infected by human T-lymphotropic virus(HTLV-1).Viral protein Tax through the regulation of multiple cell proliferation and anti-apoptosis-related genes,thereby promoting the malignant transformation of T cells into ATL cells.Fas / FasL apoptotic signaling pathway is an important way to regulate the homeostasis and function of T cell immune response.When activation of effecting T cells to complete the removal of pathogens,the Fas / FasL apoptotic signaling pathway facilitates activation of T-cell apoptosis.Fas is a transmembrane protein that belongs to the family of tumor necrosis factor receptor superfamily.When FasL binds to it,activates Fas and forms a death-induced signal complex through its death domain and intracellular adapter protein,thereby activating Caspase-8,3,7 and other apoptotic cascade signals,thereby promoting the occurrence of apoptosis.And ATL cells have a resistance to Fas / FasL apoptosis signal,and its mechanism and HTLV-1 transcriptional activator protein-mediated apoptosis of protein-c-FLIP up-regulation.C-FLIP is an anti-apoptotic regulatory protein,which is structurally similar to Caspase-8,but it does not have enzymatic activity,which binds to Fas death domain to prevent Fas / FasL apoptosis downstream signaling activation,/ FasL apoptotic signaling pathway.Sirtuin1(SIRT1)is a nicotinamide adenine dinucleotide-dependent deacetylase,which can block cell senescence and apoptosis by degrading a variety of protein substrates and promote cell proliferation.And clinical manifestations,ATL cells SIRT1 also showed high expression levels,but whether it is involved in Fas / Fas L apoptosis signal pathway regulation is still unclear.ObjectiveTo investigate the role and possible molecular mechanism of Sirtuin 1 in Fas / FasL apoptosis-resistant ATL cells,and to provide experimental data and theoretical guidance for the prevention and treatment of clinical ATL.Methods1.The effects of different concentrations of recombinant protein FasL and SIRT1 inhibitor Salermide on the proliferation and proliferation of ATL cells were detected by MTS method.2.The effect of SIRT1 inhibitor or RNAi on the apoptosis of ATL tumor cells induced by Fas / FasL was detected by Annexin V-FITC and PI double staining.3.Western Blot and RNAi technology were used to detect the levels of anti-apoptotic-related protein expression and p65 acetylation of SIRT1 and c-FLIP in ATL cells.4.The immunofluorescence staining was used to detect the co-transfection of SIRT1 expression plasmid and cherry fluorescence tax fusion expression plasmid.5.The effect of SIRT1 expression plasmid and Tax expression plasmid on co-transfection of c-FLIP gene promoter was detected by using c-FLIP gene promoter fluorescent reporter gene system.Results1.The survival rate of ATL cells was 85 ± 2.73% compared with 40 ± 2.06%survival in control Jurkat cells treated with 10 ng/ml FasL ATL cells.At the same time,the apoptosis rate of Jurkat cells reached 76.74 ± 0.6%,the apoptosis rate of ATL cells was7.05 ± 0.5%;2.Compared with control Jurkat cells,SIRT1 inhibitor Salermide 25 μM treatment of ATL cells,the survival rate decreased 25%,the apoptosis rate increased significantly.At the same time,the apoptotic rate of RNAi knocked down SIRT1 ATL cells was also significantly increased to 20 ± 1.65%;3.Compared with Jurkat cells,the expression of anti-apoptotic-related proteins SIRT1 and c-FLIP in ATL cells was significantly increased.After knocking down SIRT1,the level of p65 acetylation was decreased and the expression level of c-FLIP protein was also decreased.4.Immuno-stained green fluorescent SIRT1 and red cherry fluorescence Tax in the cell’s cytoplasm and nucleus are co-localization;5.The expression of fluorescence signal of c-FLIP gene promoter was observed in a dose-dependent manner;ConclusionSirtuin1 may promote the regulation of c-FLIP expression by interacting with Tax,thereby promoting ATL cell resistance to Fas / FasL-mediated apoptosis. |
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