The Biologic Effect Of SARI Overexpression In Acutemyeloid Leukemia Cells And Its Possible Molecular Mechanisms

Objective 1 To construct a recombined lentivirus vector mediated SARI gene overexpression and verify its overexpression and infection.2 To identify the biological influence of SARI gene overexpression on acute myeloid leukemia(AML)cell lines.3 To explore its molecular mechanism.Methods 1 Construct,package and identify the recombined lentivirus vector mediated SARI gene overexpression(completed by Shanghai Genechem Co.Ltd.)2 We would use AML cell lines with low or loss expression of SARI gene identified by qPCR in our test.After SARI gene overexpression was done,the target cell lines would be identified via Western blot and qPCR.3 To invest the biologic effect of SARI gene overexpression on AML cell lines,we apply MTT test,clone formation experiment,AnnexinV-PE/7-AAD fluorescence test,mitochondrial membrane potential test,AO stain,DAPI stain,cell cycle test.4 To explore the possible molecular mechanisms of SARI,we employed Western blot and RT-PCR methods to observe the expression levels of apoptosis related proteins,some markers about signal transduction pathway and some transcription factors.Results 1 The sequence of the recombined vector were completely correct,and the virus titer measured via fluorescence method was 5×108TU/ml.2 We decided AML cell lines Kasumi-1?HL-60?NB4 used in our test.After infected to the target cells for 72 h,we found that green fluorescence was observed in all three cell lines.The SARI expression level of the overexpresion groups increased distinctly showed by qPCR and a 29 kD protein was also detected by Western blot technique.3 MTT test showed that the cell proliferation in SARI groups of all three Kasumi-1?HL-60?NB4 cells was inhibited remarkably compared with that of two control groups(p<0.05).Clone formation experiment revealed the clone formation ability of SARI group cells of all three Kasumi-1?HL-60?NB4 cells were significantly decreased(p=0.000).DAPI stain and AO stain showed that no obvious change could be observed in SARI groups of all three Kasumi-1,HL-60 and NB4 cells and its two control groups.AnnexinV-PE/7-AAD fluorescence test via FCM showed cells in SARI groups of all three Kasumi-1,HL-60 and NB4 cells had a significant high apoptosis rate compared with that of two control groups(p<0.001),the stage of apoptosis was almost early apoptosis.Mitochondrial membrane potential test via FCM revealed cells in SARI groups of all three Kasumi-1,HL-60 and NB4 cells had a significant reduction on mitochondrial membrane potential compared with that of two control groups(p<0.05).4 Western blot showed that the expression levels of apoptosis related proteins of cells in SARI groups of all three Kasumi-1,HL-60 and NB4 cells had changed compared with its negtive control(NC)groups,for example,Bcl-2,Bcl-xl were decreased(p<0.05),Bax,Cyto C were increased(p<0.05).The expression levels of Caspase family proteins of cells in SARI groups of all three Kasumi-1,HL-60 and NB4 cells had changed compared with its NC groups.Caspase3,Caspase8,Caspase9 of cell in SARI goup of Kasumi-1 were increased to form Actived-Caspase3 and PARP cleavage,compared with its NC group,while Caspase3,Caspase8,Caspase9 of cells in SARI goups of HL-60?NB4 cells were cleaved and reduced to form Actived-Caspase3 and PARP cleavage,compared with its NC groups.5 Western bot and RT-PCR showed that the expression level of the marker proteins on PI3 K signal transduction pathway such as PI3 K,p-PI3 K,Akt,p-Akt,4EBP1,p-4EBP1,mTOR,p-mTOR,p70 reduced in cells in SARI groups of all three Kasumi-1,HL-60 and NB4 cells compared with its NC groups,while no obvious change of the expression level of the marker proteins on MAPK signal transduction pathway such as MAPK and the expression level of the marker mRNA on STAT signal transduction pathway such as STAT1 could be observed in cells in SARI groups of all three Kasumi-1,HL-60 and NB4 cells compared with its NC groups.RT-PCR showed that the expression levels of some transcription factors in m RNA level were changed,for example,MZF-1 mRNA was reduced markedly,IL-8,AML1,TWIST mRNA were reduced inordinately in cells in SARI groups of all three Kasumi-1,HL-60 and NB4 cells compared with its NC groups.Western blot showed that the expression level of some transcription factors in protein level were changed,for example,NF-?B(RelA),C-myc protein were reduced inordinately in cells in SARI groups of all three Kasumi-1,HL-60 and NB4 cells compared with its NC groups.Conclusion Lentivirus mediated SARI overexpression system was constructed successfully,and overexpression of SARI gene exhibited antitumor effect by inhibiting cell proliferation and inducing cell apoptosis,almost early apoptosis in Kasumi-1,HL-60 and NB4 cells.Its possible molecular mechanisms was that SARI might activate the extrinsic apoptotic pathway and its crosstalk pathway:the endogenous(mitochondria)apoptotic pathways,inhibit the PI3 K signal transduction pathway and inhibit the expression of some transcription factors such as MZF-1,NF-?B(RelA),IL-8,AML1,TWIST,C-myc,particularly MZF-1and NF-?B(RelA)in Kasumi-1,HL-60 and NB4 cells.