Effects Of Different Ikaros Isoforms On Cell Proliferation And Apoptosis

ObjectiveBy analyzing the different Ikaros isoforms in clinical bone marrow samples from children with acute lymphoblastic leukemia,the effects of Ikaros loss mutation(function loss)on the biological behavior of cells and the possible molecular mechanism of multidrug resistance were explored.Personalized therapeutic targets provide a theoretical basis.Methods(1)110 bone marrow samples from children diagnosed with childhood acute lymphoblastic leukemia diagnosed at Children’s Hospital of Jiangxi Province from 2013 to 2015 were collected.The collected samples were approved by parents of the children and the ethics committee of the hospital.(2)Using lymphocyte separation liquid to isolate mononuclear cells,extract total RNA,determine the concentration and purity of RNA,reverse transcribe into cDNA,determine the Ikaros subtypes by nested PCR and clone sequencing,analyze Ikaros expression.The correlation between Ikaros isoforms and other clinical indicators,and the correlation between Ikaros abnormal phenotype and prognosis were analyzed.(3)Use IK1 as the Ikaros functional representative,IK6 as Ikaros non-functional representative,and construct Ikaros functional type and non-functional expression vector in human embryonic kidney 293 T cell line according to the transfection reagents of the Lip2000 manual.In the procedure,cells were transfected with pcDNA3.1 vector,pcDNA3.1-IK1,and pcDNA3.1-IK6,respectively,and cells were collected after 48 hours for downstream experiments.Total protein was extracted from cells,IK1 and IK6 were detected by Western blot(WB),cell proliferation rate was detected by MTT assay,and the expression levels of apoptosis-related proteins Bax and Bcl-2 were detected by WB,and cell apoptosis was detected by chemiluminescence.The levels of caspase3,a relevant factor of death,were used to demonstrate the effects of IK1 and IK6 on the biological behavior of 293 T cells proliferation and apoptosis.At the same time,the expression of apoptosis-related genes(BCL-2,BAX)was quantitatively detected by qPCR fluorescence and the effects of deletion mutant Ikaros on cells were compared.To analyze the molecular mechanism of Ikaros functional status influencing the PI3K-AKT signaling pathway and the molecular mechanisms involved in apoptosis.After being compared with pubmed sequences,Ikaros primers with restriction sites(BamHI and Xho I)were designed,amplified by PCR,recovered by gel and double-digested with the expression vector pcDNA3.1 expression vector,and ligated with T4 ligase.Suspected positive recombinants were sequenced and sequenced.(4)Lentivirus packaging GV358-3FLAG-EGFP-PURO-IK6(Lenti-IK6),determination of virus titer to determine the viral load(MOI value)of human leukemia cell line Jurkat,using puromycin as a resistance screen Construct Lenti-IK6 stable cell line.Lenti-mock was used as the basic structure of viral recombinants.In the Jurkat cell line of leukemia,Lenti-IK6 and negative control packaged virus were transfected into suspension cells Jurkat using empty vector virus according to lentivirus transfection instructions.For a negative control(Lenti-Mock),a stable cell line was screened,WB was used to detect Ikaros protein expression,and used for detection of lentivirus transfection efficiency.Quantitative detection of by fluorescence was performed.The expression levels of apoptosis-related genes(BCL-2,BAX)compare the effects of deletion mutant Ikaros on cells.At the same time,the expression levels of apoptosis-related proteins Bax and Bcl-2 and the total protein and phosphorylation levels of PI3K-AKT in Lenti-Mock and Lenti-IK6 groups were detected by WB.The changes of proliferation and apoptosis levels in the two groups of cells and their effects on the PI3K-AKT signaling pathway were compared.(5)Use GraphPad prism5 software for data analysis and plotting.Routine tests for homogeneity of variance and normality tests.Measurement data experimental data is represented by.The t-test was used to compare the data between the two groups of univariate variables;the comparison between groups of data was performed using one-way analysis of variance;non-parametric tests were used for variances.The difference was statistically significant at P<0.05.Results(1)In 110 cases of ALL,the functional subtype Ikaros was dominated by Ikaros1(Ik1)and Ikaros2(Ik2),and the non-function subtype was Ikaros6(Ik6).The Ik6 expression rate was 20.91%,of which 3 cases.In children with Ik6 alone(2.73%),the heterozygous expression of functional and Ik6 was 18.18%,and the expression of Ik6 in relapsed children was increased.(2)In vitro experiments showed that the expression of non-functioning Ik6 resulted in accelerated cell proliferation and blocked apoptosis,and activated the PI3K-AKT signaling pathway,resulting in reduced sensitivity to chemotherapeutic drugs.(3)The infection index of Lenti-Ik6-infected human leukemia cell line Jurkat was 100(MOI=100),and the screening concentration of stable transformation cell line with puromycin was 2.5μg/mL.ConclusionThe non-functioning subtype Ik6 is an independent risk factor for prognosis and is closely related to the recurrence of childhood ALL.Ik6 activation of PI3K-AKT signaling pathway may be one of the mechanisms of prognosis in childhood acute lymphoblastic leukemia...