Role Of PDGFR? In Essential Thrombocythemia Induced By CALR Mutants

Objective:Myeloproliferative tumor(MPN)is a malignant hematological disease characterized by clonal proliferation of erythroid,granulocytic or megakaryotic myeloid cells.Three classical MPN diseases include primary thrombocytosis(ET),polycythemia vera(PV),and primary myelofibrosis(PMF).The clinical manifestations of these three diseases are similar and can be transformed into each other,which increases the complexity of clinical diagnosis.Essential thrombocytosis(ET)is one of the most common MPN diseases.The hematological characteristics of ET are the abnormal proliferation of megakaryocytes in bone marrow,which leads to the continuous increase of platelets in peripheral blood.Mutations in JAK2,MPL and CALR genes can lead to ET.The molecular mechanism of ET induced by JAK2 and MPL is clear,but the molecular mechanism of ET caused by CALR mutation is still unclear.Recent studies suggested that MPL-JAK2-STAT pathway can mediate the occurrence of ET.However,this mechanism can not explain the phenomena that the megakaryocytes produced by CALR mutation proliferate excessively and the number of granulocytes was basically normal.Therefore,we believe that there may be a new mechanism involved in the process of ET induced by CALR mutation.After previous work,we found that PDGFR? may be a key molecule mediating ET induced by CALR mutation.In this study,we observed the effect of CALR mutation on the proliferation of megakaryocytes before and after the inhibition of PDGFR ? activity by constructing the model of CALR mutant human megakaryocyte line,that is,UT-7/EPO,and explored the mechanism of PDGFR? mediated CALR mutation promoting the malignant proliferation of megakaryocyte line.That is to say,the function of PDGFR? was verified at the cell level.This study will help to improve the understanding of the mechanism of ET induced by CALR mutation and provide a target for accurate treatment of ET.Methods:1.Study on the effect of PDGFR? mediated CALR mutation on malignant Proliferation of megakaryocytes.UT-7/EPO cells were selected as the cell model in this study.The experimental grouping design of this cell line was: CALR type1 mutation group,CALR type2 mutation group,wild type CALR group and empty expression vector group,named CALR-MT1,CALR-MT2,CALR-WT,MSCV respectively.CALR-MT1 and CALR-MT2 were both experimental groups,CALR-WT was negative control group,MSCV was blank control group.The lentivirus recombinant expression vector p CDH1-MSCV-MCS1-T2A-cop GFP was constructed,including CALR-MT1,CALR-MT2 and CALR-WT.The expression plasmids,namely CALR-MT1,CALR-MT2,CALR-WT and MSCV,were co-transfected with packaging plasmids p PACKH1(including gag,rev,vsv)in a certain proportion by liposome 2000 into 293 T cells(within 15 generations).The expression of GFP was observed by confocal laser microscopy at 48 hours after transfection.The lentivirus suspension was collected 48 hours after transfection and filtered by 0.45 um membrane.After obtaining lentiviral particles,UT-7/EPO cells were infected with lentiviral particles in the optimum proportion.The expression of GFP was observed by confocal laser microscopy for 72 hours.The infected cell lines were expanded,GFP positive clone cells were selected by flow cytometry(FACS),the expression of target gene in the cells was detected by RT-PCR and Western bolt,and the stable cell line was established and preserved.The experiment was divided into two groups: PDGFR? non-suppressed group and PDGFR? suppressed group.PDGFR ? inhibition group was inhibited by AG-1295,a small molecular chemical inhibitor of PDGFR?.The specific methods were as follows: in the PDGFR? inhibition group,the UT-7/EPO cells were cultured in the medium containing AG-1295 inhibitor(10%FBS RPMI 1640 without EPO).Cells at different time points(0d,2d,4d,6d,8d)of each group were collected,and cell proliferation was detected by CCK-8 method;PDGFR ? uninhibitor group and PDGFR ? inhibitor group were cultured at the same time point(0d,2d,4d,6d,8d).)Cells were collected and their proliferation was detected by CCK-8 method.Each group of experiments need to be repeated three times,and the proliferation of each group before and after inhibition at the same time point was counted and compared.2.Preliminary study on the Mechanism of PDGFR ?-mediated CALR mutation promoting malignant Proliferation of megakaryocytes.The CALR mutant UT-7/EPO cells and negative control cells were collected,the protein supernatant was extracted,PBS was washed twice,and the total protein was extracted by lysing the cells.Protein quantification was carried out,and phosphorylated PI3 K protein,phosphorylated Akt protein expression level,non-phosphorylated PI3 K protein and Akt protein expression level were analyzed by Simple Western method.Results:1.In PDGFR? uninhibited group(0?mol/L),UT-7/EPO CALR-type1 and CALR-type2 cells proliferate even in the absence of EPO,whereas CALR-WT wild type group,MSCV empty vector group and UT-7/EPO cells stop proliferating,indicating that the expression of mutant CALR promotes cytokine-independent growth in human UT-7/EPO cells.However,after inhibiting PDGFR ? by 20 and 30 ? mol/L AG-1295,the proliferation of cells in each group was significantly inhibited.There was a significant difference in the degree of cell proliferation between the PDGFR? uninhibited group and the PDGFR? inhibited group at the 4th day(P < 0.05).2.There were no significant differences in phosphorylation of AKT(Thr 308),p44/p42MAPK(Erk1/2)(Thr202/Tyr 204)between CALR-type 1,CALR-type 2 and CALR-WT wild-type group in the PDGFR? non-inhibition group(0?mol/L).In the 20?mol/L AG-1295 PDGFR? inhibition group,there were no significant differences in phosphorylation of AKT and ERK in CALR-type1 and CALR-type2 compared with CALR-WT wild type group.There were no significant differences in AKT(Thr 308)phosphorylation,p44/p42 MAPK(Erk1/2)(Thr202/Tyr 204)phosphorylation between PDGFR? uninhibited group and 20?mol/ LAG-1295 PDGFR? inhibited group.Conclusion:UT-7/EPO cells with wild CALR could not proliferate normally without EPO,while UT-7/EPO cells with mutant CALR could proliferate without EPO,suggesting that PDGFR ? may be involved in the process of CALR mutation promoting cell proliferation.At the same time,AG-1295,an inhibitor of PDGFR ?,can inhibit the proliferation of UT-7/EPO cells with CALR mutation,suggesting that PDGFR ? is involved in the process of CALR mutation promoting cell proliferation,which is another mechanism of CALR mutant promoting cell proliferation.