Effects Of SIRT1 Overexpression On THP-1 Proliferation,Apoptosis And Cell Cycle And Mechanism

Objective Construction of silent mating-type information regulation 2homologue 1(sirt1)gene overexpression lentiviral vector by lentiviral vector co-transfection packaging technique,infection of acute myeloid leukemia cell line THP-1,To study the changes of biological behavior of SIRT1 ge nes on cell cycle,apoptosis and proliferation of THP-1 cells(Human acute myeloid leukemia cell line).Methods1.Identification of SIRT1 gene overexpressed lentiviral vectorWe provided the target gene sequence information,Shanghai Ji Kai company assisted in the construction of SIRT1 gene overexpressing lentiviral vector and determined the virus titer.Transfected 293 T cells were observed with green fluorescence.We used real-time quantitative RT-PCR and Western blot to identify the lentiviral vectors that were constructed at mRNA expression and protein level.2.The changes of cell proliferation,cell cycle and apoptosis after tran sfection with lentiviral vector.THP-1 cells were transfected with lentivirus with SIRT1 gene,Fluorescence detection,Rt-PCR was used to verify the expression of SIRT1 gene at mRNA level,Western blotting technique was used to verify the expression ofSIRT1 protein.CCK-8 method was used to plot the cell growth curve,Flow cytometry was used to detect the proliferation,cell cycle and apoptosis of THP-1cells after overexpression.3.Detect the expression of malignant cells in THP-1 cells after trans fectionReal-time quantitative RT-PCR detection of sirtuins family gene;Tumor suppressor gene:p21,P53;Proto-oncogene: known as leukemia-associated proto-oncogene cyclin D1(cyclinD1),Nuclear factor κB(NF-κB);Cell Proliferation / Differentiation Related Factors:Granulocyte Colony-Stimulating Factor(G-CSF),Granulocyte Colony Stimulating Factor Receptor(G-CSFR)gene,C/EBP-αand C/EBP-β;Apoptosis-related genes:B cell leukemia/lymphoma2(Bcl2),Bcl-associated X protein(Bax),cysteinyl aspartate specific proteinase caspase 3,caspase9 gene mRNA expression.The expression of activated caspase3 protein was detected by WB.4.statistical analysisThe experimental data were analyzed using SPSS 22.0 software for statistical analysis,P <0.05 was statistically significant.The data were expressed as mean ± standard deviation.Student-to-group differences were analyzed by Student’s t-test or One-Way ANOVA.The cell count at different time points was analyzed by variance analysis of the repeated measurement data of the general linear model.The data were spherical and the Huynh-Fledt condition was not used.Greenhouse-Geisser.Results1.overexpressing lentiviral vector transfected 293 t cells.Identified by RT-PCR and western blotting.Fluorescence positive.mRNA expression of SIRT1 was up-regulated more than 2-fold.The expression of target protein wassignificantly increased(p < 0.05).SIRT1 gene overexpression lentivirus was constructed successfully.2.lentivirus transfection MOI 100,successful establishment of SIRT1 gene overexpressing THP-1 cell line.RT-PCR and western blotting showed that the mRNA and target protein were significantly increased.3.SIRT1 gene overexpression lentivirus infected THP-1 cells,the cell growth curve and the control group THP-1 cells were significantly different,significantly inhibited THP-1 cell growth.proliferation index were significantly decreased;Cell proliferation index was significantly lower than that of empty virus transfected control group,P value: 0.004;Cell cycle occurred in G1 / G0 phase arrested.The apoptosis rate of THP-1 cells transfected with SIRT 1lentivirus overexpressing vector was significantly increased.4.The expression of SIRT1,SIRT3 and SIRT4 mRNA in THP-1 cells overexpressing SIRT1 gene was significantly higher than that in empty vector con145 transfection group(p <0.05);the expression of caspase3 mRNA in THP-1 cells was significantly increased,p value is 0.002.Can detect caspase3 protein expression;proto NF-κB gene downregulation,up-regulated the expression of tumor suppressor gene p21 in THP-1 cells;down-regulate the expression mRNA of BAX and Bcl2.Conclusions:SIRT1 gene overexpressed lentiviral vector can influence the expression of the gene by affecting the cell cycle.Induced cell G1 / G0 arrest,significantly inhibited cell proliferation.May affect the expression of apoptosis-related factors.Affect the apoptotic signaling pathway,induced cell apoptosis.SIRT1 gene may play an anti-cancer effect in acute myeloid leukemia.The mechanism may be related to cell proliferation,apoptosis and cycle.