Screening And Establishment Of Human Acute Myeloid Leukemia Cell Lines With Stable Interference BAALC Gene Expression

BackgroundAcute leukemia(AL) is a heterogeneous group of diseases characterized by uncontrolled proliferation of clonal neoplastic hematopoietic precursor cells as well as blockage of myeloid differentiation at different stages.Many genes and transcription factors expression belonging to signal transduction pathways,were involved in the process of leukemogenesis through affecting proliferating, differentiation,apoptosis of hematopoietic cells in molecular patterns,but the underlying mechanisms were yet unknown..With the continuous improvement of treatment, current chemotherapy can induce complete remissions in more than 80%patients,but most of them relapse.In this context,allogeneic hematopoietic stem cell transplantation(allo-HSCT) in first complete remission is generally accepted as the only curative option.In fact,for whom the probability of relapse is approximately 30%,and the transplant-related mortality is up to 20%to 40%.Therefore,refractory or/and relapse was still the most arduous problem in leukemia,which may benefit from more intensive consolidation chemotherapy regimens and allo-HSCT.However, intensive chemotherapy and allo-HSCT are still associated with high treatment-related adverse events and mortalities.Thus,it is urgently to develop novel treatment optimization in leukemia.During the past decade,biological target therapy is a focus of leukemia research,various molecular markers and global gene expression profiling have been investigated to improve the treatment outcome of leukemia patients.It highlighted in searching new prognostic molecular markers, which might be developed might develop as a potential effective treatment strategy to boost the antileukemic efficiency.Brain And Acute Leukemia,Cytoplasmic(BAALC),located on chromosome 8q22.3,as one of the most important independent risk indicators within the heterogeneous group of cytogenetically normal AML patients,predicted high minimal residual disease and inferior survival.Normally,BAALC was mainly expressed in neuroectoderm-derived tissues and hematopoietic precursor cells,whereas no or very low detection was in mature bone marrow or circulating pheriferal blood cells.DNA sequence and expression pattern were highly conserved among mammals,whereas no orthologs were found in lower organisms.The protein sequence showed no homology to any known proteins or functional domains.In leukemias,Baldus et al found that high BAALC was expressed in subsets patients with acute myeloid leukemia(AML), acute lymphoblastic leukemia(ALL),and chronic myelogenous leukemia(CML) in blast crisis,whereas no BAALC expression could be detected in patients with chronic-phase CML or chronic lymphocytic leukemia(CLL).Given the situation that BAALC expression in normal bone marrow is restricted to the compartment of progenitor cells and that it shows high expression in a subset of leukemic blasts, BAALC may be seen as a stage-specific marker regulated during hematopoiesis and aberrantly expressed in leukemogenesis.The function of the BAALC protein in hematopoiesis and leukemogenesis contributing to a more aggressive behavior of AML has yet to be identified.Bienz et al reported that about 70%of normal cytogenetics AML(NC-AML) patients highly expressed BAALC gene,and the BAALC expression was closely related to prognosi.Higher BALLC expression,the worse prognosis.Baldus et al also reported high BAALC expression in conjunction with FLT3 mutation status successfully identifies high-risk patients within the heterogeneous group of NC-AML patients.High BAALC expression is established as one of the most important independent risk factors associated with resistant disease,a high CIR,and inferior survival,disease-free survival(DFS) and overall survival(OS) was significantly shorter.High BAALC expression remained a significant adverse prognostic factor for FLT3-ITD WT and MLL-TD patients,compared to those with low BAALC expression(hazard ratio 2.7),even when the 8 additional patients harboring a FLT3/ITD genotype with normal cytogenetics and treated on CALGB 9621 were included in the multivariable analyses for OS and DFS.Our Preparatory work by using the real-time quantitative PCR(RQ-PCR) to detect AML patients BAALC gene expression,also found that the majority of newly diagnosed AML can occur BAALC high expression,while the normal control group were not detected,CR rate was significantly lower of high BAALC expression AML than those with low expression,OS shorter suggesting it might develop into a risk stratification of AML new target.Because of the infancy in BAALC research,the current understanding of BAALC was limited The functions of BAALC gene and protein,molecular mechnism,network and transduction signal pathway,in neuroectodermderived tissues and tumors,hematopoietic progenitor cells in leukemic blasts were yet unclear,all of it remains to be explained.Long time ago,the study of gene function mainly relied on gene knock-out and antisense nucleic acid technology and other methods.However,many issues,such as long cycle,complex operation and specificity,limited the use of them in large-scale study of gene function.In recent years,some small double-stranded RNA could be highly efficient and specific blocking of certain genes in vivo resulting in mRNA degradation,inducing cell-specific gene deletion and showing some phenotype, named RNA interference(RNAi).The main advantage of RNAi was reverse genetic analysis,a specific gene sequence is known,through the inhibition of the expression of specific genes and individual functional and phenotypic changes,which have been genetically linked to their functional counterparts.In addition,the technology also was specific,efficient,fast and cutting of the target RNA,high stability and high penetrating characteristics.Since Andrew Fire and Craig Mello showed that double-stranded RNA molecules could inhibit the expression of homologous genes, RNAi as the greatest biological discovery,brought a revolutionary change in the life medica science research during the the past decade in gene expression,successful applications in animal models have already led to the initiation of RNAi-based clinical trials as a new therapeutic option.ObjectiveTo Screen and establish human acute myeloid leukemia kasumi-1 cell line with stable silencing BAALC(brain and acute leukemia,cytoplasmic) gene expression by RNA interference(RNAi).MethodsFour pairs of oligonucleotide sequences specific targeting BAALC gene were designed and synthesized,and cloned into pGPU6/GFP/Neo plasmid vector.The recombinant plasmids were amplified in Ecoli.DH-5αwas identified by restriction digestion and sequencing.The vectors were transfected into kasumi-1 leukemia cell line by lipofectamine2000 and the cells stably expressing shRNA were selected with G418.Effect of interference in mRNA and protein level was detected by real-time RT-PCR and Western blot analysis.ResultsBeen annealed shRNA by 100bp Department agarose gel electrophoresis a single electrophoretic bands can be seen that the success of Oligo single strand annealing. Empty plasmid pGPU6/GFP/Neo was digested by BamHⅠand BbsⅠrestriction endonuclease,37℃overnight,digestion product was agarose gel electrophoresis,it can be seen and the 5kb and 57bp bands on plasmid extraction.5k b product was purified by the Gel can be used to connect the next step reaction.The purification product was connected with the annealing product of E.coli after transformation into DH-5α,blue-spot screening of positive clones.In order to determine whether the shRNA expression plasmids constructed,recombinant plasmid DNA was extracted and confirmed by sequencing,sequence primer were as follows: 5'-GGACTATCATATGCTTACCG-3' The recombinant plasmids vector were obtained successfully.After transfection 48h,cells were collected to observe GFP expression by fluorescence microscope.It could see some cells were GFP-positive cells collected 48h transfection.After washing by PBS,cells were collected to detection of GFP expression by flow cytometry(FACS) and transfection efficiency was about 5%.To determine whether the shRNA expression vectors targeting BAALC could down-regulate gene and inhibite protein expression,the silence effects in mRNA and protein levels were assayed by real-time RT-PCR and Western blotting.The leukemia cell line exhibited overexpression of BAALC mRNA when compared to the stably transfected cell line.The level of BAALC mRNA was decreased by 61%and 74%in cells transfected by pshRNA-BAALC1～4,respectively.However,no change was observed in cell lines transfected by the nonspecific control groups.The expression of BAALC protein was declined by the two shRNA expression vectors.The levels of BAALC were decreased by 40%and 62%in cells transfected by pshRNA-BAALC1～4,respectively.Cells transfected by negative controls did not show any changes.The interference effect in protein level was in accordance with the mRNA level.The stably silencing cell lines were established successfully.ConclusionThe shRNA plasmid expression vector targeting BAALC gene was succesfully constructed and stable silencing cell line was obtained,which provides a basis for further study the relationship between leukemia and BAALC gene.