Micrornas The Aml1a Role In The Abnormal Differentiation And Malignant Transformation Of Hematopoietic Cells In Mice And Atra Treatment Of Nb4 Cells And Expression Analysis

PartⅠThe role of AML1a in abnormal proliferation and differentiation and its significant function in malignant transformation of murine hematopoietic cellsCell proliferation and differentiation are strictly regulated by intrinsic and extrinsic signals,in which coordination of a variety of gene expression by transcription factors is the most important mechanism.Uncontrolled proliferation and impaired differentiation of hematopoietic cells lead to leukemogenesis.Therefore,it is significant to clarify the functions of transcription factors for elucidating the mechanism of abnormal proliferation and differentiation,as well as leukemogenesis.AML1,also called RUNX1,PEBP2αB or CBFα2,is a critical transcription factor in hematopoietic cell differentiation and proliferation.Its mutation and translocation lead to hematopoietic malignant.From the AML1/RUNX1 gene,at least three isoforms,AML1a,AML1b and AML1c,are produced through alternative splicing.The proteins encoded by AML1b and AML1c have a RHD in the N-terminus and a transactivation domain in the C-terminus,which are often called AML1.In contrast,the protein encoded by AML1a has a RHD but lacks the transactivation domain.As such,the function of AML1 is believed to be mediated by AML1b and AML1c which are considered to have the same function.AML1a interferes with the function of AML1b/1c.It has been proved that AML1a is higher expressed in acute myeloid leukemia(AML) than in normal controls.Our previous study confirmed that AML1a expression in acute leukemia(AL) has a high level.In addition,AML1a can inhibit the transcription of M-CSFR promoter mediated by AML1b,suggesting that AML1a has the effect of antagonism to AML1b.Some scholars believe that loss of the transcription activation domain may cause leukemogenesis,but at present there is no report in this regard.In order to explore the role and mechanism of AML1a in abnormal hematopoesis and leukemogenesis,we completed the following work in this study:1.The construction of AML1a expression vector and the production of retrovirus.pMSCV-FLAG-AML1a-IRES-YFP was constructed.293T cells were transfected with pMSCV-IRES-YFP,pMSCV-FLAG-AML1a-IRES-YFP,the envelope-encoding plasmid pV Pack-Eco,or the packaging plasmid pV Pack-GP using a method of calcium phosphate precipitation to product retrovirus.The title of virus was measured using a bioassay with the 3T3 cell line.The expression of the fusion proteins in the tranduced cells was confirmed.The title of retrovirus can satisfy the continually experimental demand.2.The role of AML1a in abnormal hematopoiesis and leukemogenesis.1) Bone marrow mononuclear cells(BMMNCs) were infected with the retroviral vector MSCV expressing a FLAG-AML1a fusion protein and a yellow fluorescent protein(YFP).The cells were cultured in Iscove modified Dulbecco medium(IMDM) supplemented with murine IL-3(mIL-3),murine IL-6(mIL-6) and marine SCF (mSCF).The change of surface marker of each hematopoietic linage was analyzed by FACS in 11d,23d and 35d after transduction.The result showed that the expression of Sca-1 and Thy1.2 in AML1a transduced BMMNC was higher than that of the control,which indicating that AML1a may prone to block the differentiation of hematopoietic stem cell in the relatively early lymphocytic stage 2) BMMNCs from mice were transduced with AML1a and transplanted into lethally irradiated mice, which develop lymphoblastic leukemia after transplantation.Lethally irradiated female C57 mice received BMCs infected with the retroviral vector MSCV expressing a FLAG-AML1a fusion protein and a yellow fluorescent protein(YFP). BMCs infected with the vector expressing YFP only was used as a control,Number of YFP-positive cells in the peripheral blood as well as general health condition of the mice was monitored.9 out a total of 12 mice in the AML1a group developed leukemia at 3-11 months.Autopsy of these mice revealed spleenon(?)agly and hepatomegaly with thymoma and lymph node enlargement.Bone marrow,spleen, lung,liver,kidney and thymus were infiltrated with leukemia cells.A further analysis revealed that the BMCs in mice with leukemia mainly exhibited 2 phenotypes.One was Sca-1~+ cytoplasmic CD3~+(cCD3~+),a common type of T-lymphoblastic leukemia. The other was CD3~+CD4~+CD8~+,a type of T lymphocytic leukemia.All mice received secondary transplantation rapidly developed lymphocytic leukemia.Taken together, these results indicate that overexpression of AML1a may be an important contributing factor to leukemogenesis.3) Cell cycle of BMCs from the secondary recipient of leukemia cells was examined.Representative cycle distribution of the BMCs was shown that BMCs in the AML1a group obviously were of higher percentage in G0/G1 phase and lower percentage in S phase in comparison to the control group,which indicated that cell cycle of transduced AML1a hematopoietic cells was arrested in the G0/G1 phase AML1a may alter the process of normal cell proliferation and division and lead to the malignant transformation of hemotopoietic cells.In conclusion,we transduced AML1a into BMMNC of mice by retrovirus and transplanted these cells into lethally-irretiated mice.As a result,this study has revealed,for the first time,a new effect of AML1a in leukemogenesis.It indicates that AML1a may play a critical role in leukemogenesis,especially in the development of lymphoid leukemia.In addition,lymphoblastic leukemia model established in this study may serve as a valuable tool for future studies.These results may also have implications in the treatment of leukemia. PartⅡAnalysis of microRNAs expression profiles of acute promyelocytic leukemia cell line NB4 treated with all-trans retinoic acidMicroRNAs(miRNAs) are an abundant class of small non-protein-coding RNAs that function as negative gene regulators to regulate diverse biological processes. Each miRNA can control hundreds of gene targets.Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers and indicates that miRNAs can function as tumour suppressors and oncogenes.Thus, miRNAs might prove useful in the diagnosis and treatment of cancer.To explore the profile of microRNAs in acute promyelocytic leukemia(APL) cell line NB4 treated with all-trans retinoic acid(ATRA) and to look for the microRNAs which affect on cell differentiation.The differentiation of NB4 cell treated with ATRA was verified by cell morphology and flow cytometry.The difference of microRNAs expression profiles of NB4 cells treated with ATRA at different time was analyzed by means of microRNAs array.Real-time RT-PCR was used to testify the change of microRNAs expression,and the target genes of microRNAs were predicted.NB4 cells can be induced to terminal differentiation after 3 days exposure to ATRA and the differentiation antigen CD11b increased significantly from 3%to 96%.Using microRNAs array,24 differentially expressed microRNAs were screened from 576 human microRNAs candidates in which,21 microRNAs were up-regulated and 3 down-regulated.Further more,by microRNA-target gene prediction assay,the predicted target genes of the 16 up-regulated microRNAs were in volved in leukemogenesis related HOX genes.The expression of the two most significantly up-regulated microRNAs(miR-210 and miR-342) was confirmed by real time RT-PCR.The ratio of the two microRNAs after normalization was 2.16 and 4.61 respectively.The differentially expressed microRNAs in ATRA treated NB4 cells may be associated with the differentiation.Furthermore,the change of microRNAs expression may provide basis for the control mode of repressing gene expression at post-transcription level.