The Study Of Inducing Cycle Arrest And Apoptosis Of Leukemia Cells By Using Physcion Down-regulated HOXA5

Objective:The aim of this study is to investigate the effects of Physcion on the expression of HOXA5 genes in human acute lymphoblastic leukemia(ALL)cell lines NALM6 and SUPB15 in vitro,and to investigate the effects of Physcion on cell cycle and cell apoptosis and provide the experimental basis for the treatment of ALL by using Physcion.Methods:1.Cell culture:Human ALL cell lines are cultured by applying in vitro cell culture techniques.2.Experimental grouping:Two human ALL cell lines,NALM6 and SUPB15 are selected for the experiment.(1)Firstly,the cell lines NALM6 and SUPB15 are respectively divided into three groups according to the different concentrations of Physcion(0μM,2μM,5μM);(2)After the HOXA5 genes are down-regulated by siRNA,NALM6 and SUPB15 cells are divided into four group:the blank control group,the siRNA intervention group,the Physcion intervention group,and the siRNA+Physcion intervention group;(3)After HOXA5 genes become overexpressed,NALM6 and SUPB15 cells are divided into four groups:the blank control group,the HOXA5 genes overexpression group,the Physcion intervention group,and the HOXA5 genes overexpression+Physcion intervention group.3.Cell counting kit-8(CCK8)detection:(1)The effects of different concentrations of Physcion on cell proliferation after 24h,48h,72h,96h are detected;(2)At the same time,the best intervention concentration and intervention time of Physcion are determined;(3)Detect the cell viability in each group at various time points after transfecting HOXA5-targeted siRNA.4.Quantitative real-time PCR(qRT-PCR)detection:The expression of mRNA of HOXA5 genes in each group is detected 48h after the intervention by using Physcion of different concentrations.5.Western blot detection:(1)The protein expression of HOXA5 genes in e ach group of cells is detected after 48-hour intervention by using Physcion of different concentrations;(2)Detect the protein expression of HOXA5 genes targeted by siRNA and overexpressed plasmids of HOXA5 genes in each group;(3)Detect the protein expression of cleaved activated caspase-3,cleaved activated poly ADP-ribose polymerase(PARP),other apoptosis-related genes B cell lymphoma/leukemia-2(Bcl-2),Bax,as well as Cycle-related genes Cyclin D1.6.[3H]-thymidine ribotide([~3H]-TdR)detection:To evaluate the effect of inhibited expression and overexpression of HOXA5 genes by using Physcion and targeted siRNA on cell proliferation.7.Cell apoptosis detection:(1)To detect the apoptosis of two ALL cell lines after the intervention by using Physcion of different concentrations at various time points;(2)After two ALL cell lines are transfected by HOXA5-targeted siRNA and overexpressed plasmids of HOXA5 genes,the cell apoptosis of each group is detected.8.Cell cycle detection:(1)Detect the cell cycle of two ALL cell lines a certain period of time after intervention by using Physcion of different concentrations;(2)After two ALL cell lines are transfected by HOXA5-targeted siRNA and overexpressed plasmids of HOXA5 genes,the cell cycle of each group is detected.Results:1.Physcion inhibits cell growth in ALL cells by inducing cell apoptosis and cell cycle arrest.(1)After these two kinds of ALL cell lines,NALM6 and SUPB15,are intervened by using Physcion,the cell viability decreases in a dose-and time-dependent manner;(2)[3H]-TdR incorporation assay shows that Physcion inhibits the proliferation of the ALL cells in a dose-dependent manner;(3)Physcion induces apoptosis of ALL cells in a dose-dependent manner;(4)After the Physcion intervention,the caspase-3 and PARP in ALL cells are dose-dependently activated;(5)Physcion induces G1 arrest of ALL cell cycle in a dose-dependent manner.2.Physcion exerts its anti-leukemia cell effect by down-regulating the expression of HOXA5 genes.(1)Physcion inhibits the mRNA expression of HOXA5 genes in a dose-dependent manner;(2)Physcion inhibits the protein expression of HOXA5 genes in a dose-dependent manner;(3)Physcion is involved in the regulation of ALL cell cycle and cell apoptosis by inhibiting the expression of the HOXA5 genes.3.HOXA5 genes is the main target of anti-leukemic cells in vitro.3.1 Physcion inhibits ALL cell proliferation by regulating HOXA5 genes.(1)Overexpressed plasmids of HOXA5 genes significantly enhances the protein expression of HOXA5 genes;(2)siRNA targeting HOXA5 genes significantly inhibits the protein expression of the HOXA5 genes;(3)The overexpression of HOXA5 genes significantly reduces the inhibitory effect of Physcion on ALL cell growth;(4)siRNA targeting HOXA5 genes can enhance the inhibitory effect of Physcion on cell growth;(5)[3H]-TdR incorporation assay detects the effect of Physcion on the proliferation of ALL cells by regulating HOXA5 genes.3.2 HOXA5 genes mediates the induction of cell apoptosis by using Physcion.(1)The overexpression of HOXA5 genes reduces the apoptosis-inducing effect of Physcion;(2)siRNA targeting HOXA5 genes promotes Physcion-induced apoptosis in ALL cells;(3)The overexpression of HOXA5genes decreases the activation of caspase-3 and PARP and the expression of apoptosis-related genes when regulated by Physcion;(4)siRNA targeting HOXA5 genes can enhance the activation of caspase-3 and PARP induced by Physcion and its effect on apoptosis-related genes.3.3 Physcion arrests the cycle of ALL cells through targeted down-regulation of HOXA5 genes.(1)The overexpression of HOXA5 genes increases the number of S-phase cells and attenuates the cell cycle arrest in G1 phase;(2)siRNA targeting HOXA5 genes significantly enhance cell cycle arrest in G1 phase;(3)the overexpression of HOXA5 genes enhances Cyclin D1 expression;(4)siRNA targeting HOXA5 genes decreases Cyclin D1 expression.Conclusion:1.Physcion inhibits cell growth by inducing apoptosis and cell cycle arrest in the human ALL cell lines,NALM6 and SUPB15.2.The overexpression of HOXA5 genes obviously eliminates the inducing effect of Physcion on apoptosis and cell cycle arrest of ALL cells.In contrast,knockdown of HOXA5 genes by siRNA enhances the inhibitory effect of Physcion on ALL cell lines.