Study On The Sensitivity Of E5(-)E6(-) Spliceosome Of L-type Pml-rarα Fusion Gene To As2O3

Objective:Because of L type PML-RARα fusion gene of acute promyelocytic leukemia the PML gene have a alternative splicing,there is forming three different transcription splicing,E5(-)E6(+)(lack of exon 5),E5(-)E6(-)(5、6 exons are missing),E5(+)E6(+)(5、6 exons are present).Previous studies have confirmed that acute promyelocytic leukemia patients with high expression of E5(-)E6(-)spliceosome have a poor prognosis,perhaps due to its loss of nuclear localization signal leads to cytoplasmic localization and showed a poor sensitivity to ATRA induced differentiation.This study was to investigate the sensitivity of E5(-)E6(-)spliceosome to As2O3 at a cellular level of clonal cells which stablely expressed splicing bodies and NB4 cells,so as to provide treatment guidance for APL(acute promyelocytic leukemia)clinical patients.Methods:1.To judge the expression of CD33 、CD11b and the positive rate of Annexin-V 、PI(Propidium Iodide)by means of FCM(flow cytometry)during 0-96 h at different time points,NB4 cells were treated with 0.1μM、0.2μM、1.0μM、1.5μM and 2.0μM ATO respectively.Then,the differentiation and apoptosis were analyzed to determine the concentration of ATO.2.To detect the expression of the three spliceosomes of NB4 cells during differentiation and apoptosis of the best concentration of ATO treatment,FQ-RT-PCRwere performed.3.Recombinant lentiviral vectors p CDH1 empty vector、p CDH1E5(-)E6(+)、pCDH1E5(-)E6(-)which were pre-bulit success were co-transfected into 293 T cells withthe packaging-supporting plasmids p VSV-G、p PACKH1-REV、p PACKH1-GAG,respectively.After collecting virus particles,HL-60 cells were infected with virus particles,and the limited dilution method was used to screen different slice stable clonal cell lines.4.To judge the expression of CD33 、CD11b and the positive rate of Annexin-V 、PI(Propidium Iodide)by means of FCM(flow cytometry)during 0-96 h at different time points,stable clonal cells were treated with 0.2μM、1.5μM ATO respectively.To detect the expression of the spliceosomes of stable clonal cells during differentiation and apoptosis of the best concentration of ATO treatment,FQ-RT-PCR were performed.The changes of PML nucleus in stable clonal cells were observed by confocal immunofluorescence.Results:1.NB4 cells were treated with different concentrations of ATO,the expression of CD11 b in 0.2μM ATO group significantly higher than that in other groups with prolonging the drug action time.1.5μM ATO effect on NB4 cell,the positive rate of Annexin-V and PI with the extension of the duration of drug action is significantly increased.The optimal concentrations of ATO for differentiation and apoptosis were 0.2μM and 1.5μM,respectively.2.Results from FQ-RT-PCR showed that the expression of isoforms E6(+)、E5(-)E6(-)with the prolonged drug action declined gradually,when 1.5μM ATO act on NB4 cells,72 h and 96 h have significant difference ﹙ P<0.05 ﹚ compared with the control group;0.2μM ATO was acting on NB4 cells,with drug action time prolonged E6(+)、E5(-)E6(-)isoforms expression downward trend is not obvious,as compared with the control group no significant difference(P>0.05)at each time point;There was no significant difference in the expression of E6(+)and E5(-)E6(-)in the same concentration and at the same time.3.Screening of clonal cell stablely expressed splicing: HL-60 cells infected by lentivirus,limited dilution method used for screening clonal cell lines,HL-60-pCDH1 empty vector、HL-60-pCDH1-E5(-)E6(+)、HL-60-pCDH1-E5(-)E6(-)cell clone which stablely expressed the spliceosome were finally got,and proved byRT-PCR have the correct expression of target gene.4.Clonal cells stably expressing the splice were treated with 0.2μM、1.5μM ATO,the expression of CD11 b in 0.2μM ATO group of pCDH1E5(-)E6(+)、pCDH1E5(-)E6(-)stable clonal cells have an upward tendency with the time of drug action,the upward trend of pCDH1 empty body is more obvious;1.5μM ATO effect on stable clonal cell,the positive rate of Annexin-V and PI with the extension of the duration of drug action is increased,the upward trend of pCDH1 empty body is more obvious.Results from FQ-RT-PCR showed that the expression of isoforms E5(-)E6(+)、 E5(-)E6(-)with the prolonged drug action declined gradually,when 1.5μM ATO act on pCDH1E5(-)E6(+)、pCDH1E5(-)E6(-)stable clonal cells respectively;0.2μM ATO was acting on pCDH1E5(-)E6(+)、pCDH1E5(-)E6(-)stable clonal cells,with drug action time prolonged E6(+)、 E5(-)E6(-)isoforms expression downward trend is not obvious.There was no significant difference in the expression of E6(+)and E5(-)E6(-)in the same concentration and at the same time.5.The results of confocal immunofluorescence showed that the structure of pCDH1E5(-)E6(+)、 pCDH1E5(-)E6(-)stable clonal cells PML nucleus restored into normal spotted structure in the 1.5μM ATO group with the prolonged drug action;The structure in the 0.2μM ATO group of pCDH1E5(-)E6(+)、pCDH1E5(-)E6(-)stable clonal cells PML nucleus has no obvious change with the prolonged drug action.Conclusion:E5(-)E6(-)splicing was sensitive to ATO from endogenous and exogenous both;E5(-)E6(-)splice has a negative effect on the PML nucleus,and ATO could restore the structure of PML nucleus to normal.