The Effect Of A Novel Mutation Gene MPL A497-L498ins4 On The Proliferation Of BaF3 Cell

Objective:Myeloproliferative neoplasms(MPN)is a clonal hematopoietic stem cell disease characterized by the disorderly proliferation of one or more myeloid cells,including erythroid,granulocyte and megakable.The classic three types of MPN diseases include polycythemia(PV),primary thrombocytosis(ET),primary myelofibrosis(PMF),their clinical manifestations are more similar and can be transformed into each other.Although JAK2 V617 F,JAK2 exon12,MPL exon10 and CALR exon9 were found to be the main molecular causes of PV,ET and PMF in MPN,some patients did not find the above molecular causes.We identified a new type of mutation(named MPL A497-L498ins4)in which the MPL gene was present in two of the 771 patients diagnosed with MPN.MPL gene encoding thrombopoietin receptor(TPOR)protein molecules,TPOR transmembrane region aa514-518 when TPOR absence of ligand binding they can prevent the receptor dimer close to each other,and then preventing activation of its downstream signaling pathways such as JAK2-STAT signaling pathway to inhibit cell proliferation.The mutations of MPL gene reported in the previous study were mainly MPL W515 mutation,and it was confirmed that the mutation could lead to the ability of cells to promote spontaneous proliferation.The mechanism may be due to the abnormal structure of TPOR aa514-518 regional.MPL A497-L498ins4 mutation,although inserted into 4 amino acids,but not in the TPOR aa514-518 region,So whether the mutation is the driving gene of the patient MPN ?To this end,our group proposed by lentivirus gene transduction technology to transduction the mutate gene and control genes into mouse original B lymphocytes(BaF3),To explore the role of MPL A497-L498ins4 in the pathogenesis of autonomouslypromoting cells from the cytological level,and to provide a basis for the study of subsequent animal function of the mutations.Methods:1.Construction of lentiviral expression vectors of MPL A497-L498ins4 and controlsUsing RT-PCR to obtain the full-length CDS regions of MPL A497-L498ins4 、MPL-W515L、 MPL-WT genes.The lentiviral expression vector,which can be stably expressed in BaF3 cells,was constructed by the PCR products and MSCV which were obtained by double enzyme digestion,enzymatic hydrolysis product recovery and ligation,We identified as MPL A497-L498ins4-MSCV,MPL-W515L-MSCV,MPL-WT-MSCV recombinant plasmids.2.Obtain BaF3 cell lines which can stable expression of target gene and control genesEach genes were transduced into 293 T packaging cells by liposome,after lentiviruses were obtained,Ba F3 were infected at the right proportion with these lentiviruses.Through flow cytometry sorting,the infection rate of BaF3 cells was up to 90%.After the sorting,we expand the culture to obtain Ba F3 cell lines which can stable expression of target gene and control genes.3.Comparative analysis the effect of MPL A497-L498ins4 mutation on proliferation of BaF3 independent of IL-3Normally,BaF3 cells need to rely on IL-3 stimulation to maintain growth,after removal of IL-3,we can be better observed whether MPL A497-L498ins4 mutation can promote the proliferation of BaF3.Therefore,our group decided to taking MPL A497-L498ins4-BaF3 as the research object,MPLW515L-BaF3,MPL WT-BaF3 and MSCV-BaF3 were used as positive control,negative control and blank control.After removal of IL-3,the number of BaF3 cells in each experimental group was determined by CCK-8 at 0h,24 h,48h and 72 h to confirm whether MPL A497-L498ins4 mutation can effect cell proliferation.4.Comparative analysis of Sensitivity of MPL A497-L498ins4 Mutant to Ligand TPOThe MPL gene expresses the TPO receptor and its ligand is TPO.MPL gene mutations can cause increased autonomy,in the absence of or low concentrations of TPO can cause significant growth in cells,while the wild type MPL is not obvious.Therefore,the TPO sensitivity assay can reflect the proliferation activity of MPL A497-L498ins4 mutation.Therefore,this study can be used to determine whether the MPL A497-L498ins4 mutation has the independent activity of promoting cell proliferation by detecting the TPO sensitivity test.The experiment designs the groups as above.The number of BaF3 cells in each experimental group were measured with CCK-8 at 96 hours after culture in different TPO concentrations(2ng/ml、1ng/ml、0.1ng/ml、0.01ng/m、l0.001ng/ml、0ng/ml)to confirm whether MPL A497-L498ins4 mutation can effect cell TPO sensitivity.Results:1.Construction and identification of lentiviral vector of each genesThe MPL A497-L498ins4,MPL W515 L and MPL WT were successfully ligated into the lentivirus expression vector pCDH-MCS-T2A-copGFP-MSCV.The genes of the inserted vector were identified by the first generation of sequencing.The results were test by Blast analysis showed that all sequences were correct insertion the vector.2.Establishment and identification of BaF3 cell models which transport target gene and control genesUsing the constructed expression vectors to prepared the lentiviral suspension of MPL A497-L498ins4 and control(positive control MPL W515 L,wild type MPL WT,empty vector MSCV)and infected with BaF3 cell line.The infection rate of each group was : 82%,77%,70.5%,95.7%.After the flow splitter the positive rate of GFP in each group were more than 90%.RT-PCR and CD110-PE flow cytometry were used to identify the mRNA and protein expression of cell transduction genes in each group.The results showed that all the groups had the target genes expression except BaF3 group and the empty vector MSCV group,and the protein molecules located on the BaF3 cell membrane.All results confirm the successful establishment of a BaF3 cell models that stably expresseach genes.3.Analysis the effect of MPL A497-L498ins4 on the proliferation of BaF3 cells without IL-3 in mediumThe results showed that there was no significant change in the proliferation of MPL WT group,MSCV group and Ba F3 group at each time point,and there was no significant difference between each two groups(P> 0.05).MPL A497-L498ins4 group and MPL W515 L group showed significant proliferation at 48 h and 72 h,and there was significant difference compared with MPL WT,MSCV and BaF3 groups(P <0.01),but MPL A497-L498ins4 group was no significant difference with MPL W515 L in cell proliferation(P> 0.05).4.Analysis the sensitivity of MPL A497-L498ins4 mutant to its ligand TPOThe results showed that MPL A497-L498ins4,MPL W515 L and MPL WT had significant proliferation at 2ng/ml and 1ng/ml TPO,which were significantly different from those of MSCV and BaF3(P <0.01).MPL WT,MSCV and BaF3 had no obvious proliferative activity at other TPO,and MPL A497-L498ins4 and MPL W515 L cells proliferated significantly compared with MPL WT,MSCV and BaF3(P <0.01).Conclusions:MPL A497-L498ins4 mutants can cause BaF3 cells to be independent of IL-3 growth,and this mutant is sensitive to low-concentration TPO than wild-type MPL,suggesting that MPL A497-L498ins4 mutants have the same autonomous cell proliferation activity as MPL W515 L mutants.