| Objective:BCR-ABL negative myeloproliferative neoplasms mainly includePolycythemia Vera(PV),Essential Thrombocythemia(ET)and primary myelofibrosis(PMF).It is a hematopoietic stem cell disease characterized by malignant proliferation of one or more myeloid lineages.Mutations in JAK2V617F,JAK2 exon12,MPL W515K/L and CALR exon 9 genes have been confirmed to be the main driver mutations of MPN and can be detected in about 90%of MPN patients.However,there are about 15%ET and less than 10%PMF patients lack of these driver mutations,defined as"triple negative"MPN.In order to further explore the disease-causing factors of triple negative MPN,we screened exon 10 of MPL gene in clinical MPN patients.We found two atypical MPL mutations in three triple negative MPN patients including an insertion mutation MPL A497-L498ins4 and a double-point mutation MPLW515RQ516E.To investigate whether these two atypical MPL mutations are a direct cause of MPN,we transduced the mutation gene into Ba/F3 cells by lentivirus transduction and then constructed the cell models which can express mutation genes stably.In this study,we aimed to identify the function of two novel MPL mutations in cellular level.We hope our research can provide a supplement for the pathogenesis,diagnosis and treatment of triple negative MPNs.Method1.Screening and identification mutations on MPL exon 10 in triple negative MPNsWe used PCR to amplify MPL exon10 gene in newly diagnosed triple negative MPNs patients,PCR products was sequenced by Huada Gene Company.We blast the MPL sequence with the gene bank then there were two atypical MPL gene mutations observed,MPL A497-L498ins4 and a double-point mutation MPLW515RQ516E.We performed TA-clone analysis to determine whether the two mutation sites were located in the same DNA chain.2.Construction of lentivirus expression vectors of MPL A497-L498ins4,MPLW515RQ516E and other control genes.RT-PCR to obtain the cDNA sequence of MPLA497-L498ins4,W515RQ516E and other control genes.We design specific primers to amplify the full-length coding sequence(CDS)of MPL.PCR products were digestion by double restriction endonucleaseandligatedtothelentivirusexpressionvector pCDH-MCS-T2A-copGFP-MSCV.After the target gene was successfully inserted into the lentivirus expression vector,the sequence of each target gene was identified by sanger sequencing.3.Construction of MPLQ516E lentivirus expression vector by site-directed mutagenesis.Inorder to identify the function of single site MPLQ516E mutation,we perfomed the site-directed mutagenesis experiment.Primers was designedaccording to the requirements of QuikChange Lightning Site-Directed Mutagenesis Kit,which contained the mutation bases.The wild type MPL lentivirus expression vector was used as template for the following site-directed mutagenesis PCR reaction.DpnⅠwas used to digested PCR products in order to degrade template plasmids.We obtain the newly vectors after transforming the post-digested PCR product to DH-5αcompetent cells.Clones were selected for small amount of plasmid extraction,and sequenced.4.Construction of Ba/F3 Cell Models stably expressing MPLA497-L498ins4,MPLW515RQ516E,MPLQ516E,MPLW515L,MPLQ516E,MPL WT and MSCV.293T cells were co-transfected with lentivirus expression vectors MPL A497-L498ins4,MPLW515RQ516E,MPLQ516E,MPLW515L,MPL WT and MSCV in a certain proportion with packaging plasmid,respectively.After 48 hours,lentivirus particles were collected.Ba/F3 cells were infected according to the optimum proportion.After 72hours,the infection efficiency of each group was detected by flow cytometry.Cells with less than 90%positive rate were sorted by flow cytometry.5.Effect of MPLA497-L498ins4 and MPLW515RQ516E mutations on the proliferation of Ba/F3 cells without IL-3IL-3 factor is a necessary cytokine for Ba/F3 cells to maintain normal growth.MPLW515L mutation can cause the cytokine-independent growth on Ba/F3 cells.In this study,we performed the IL-3 withdraw test to observe the function of atypical MPL A497-L498ins4 and W515RQ516E mutations in vitro.Cells expressing MPLW515L mutation were used as positive control,and the cells expressing MPLWT were used as negative control,and vector control and untransfected Ba/F3 blank control were also set up.the proliferation of cells in each group was detected by CCK-8 assay,at 0 h,24h,48 h and 72 h,and statistical analysis was carried out.6.TPO sensitivity test of MPLA497-L498ins4 and MPLW515RQ516E mutationsTPO is the specific ligand of thrombopoietin receptor encoded by MPL.MPL gene mutation often leads to high sensitivity or independence to ligand TPO while the wild type must rely on a certain concentration of TPO to maintain cell growth.The aim of this study was to verify the sensitivity of MPLA497-L498ins4 and MPLW515RQ516E mutations to TPO.Cells in each group were cultured for 96 hours under six different concentration of TPO(2ng/ml,1ng/ml,0.1ng/ml,0.01ng/m,0.001ng/ml,0ng/ml)and then the cell counts in each group was measured by CCK-8 assay and statistically analysis was done.7.Analysis of G1,G2 and S phases of MPL A497-L498ins4 and MPLW515RQ516E mutants in different concentrations of TPO.In order to further explore the effect of mutants on cell cycle,cells in each group were cultured in different concentrations of TPO(0ng/ml,0.01ng/ml,10 ng/ml)for 48 hours.10~6cells in each group were collected.Cells conjugated with PI fluorescence(Beyotime,China)were measured using flow cytometry after fixed in 70%ethanol at 4℃overnight.8.Effect of MPLQ516E single point mutation on proliferation of Ba/F3 cells.(1)the effect of MPLQ516E single point mutation on the proliferation of Ba/F3 cells independent of IL-3.In order to investigate whether single point mutation Q516E will affect the function of double point mutation MPLW515RQ516E,we constructed a cell model to stably express Q516E mutation.IL-3 withdrawal test was performed to investigate the effect of MPLQ516E mutation on cell proliferation.(2)TPO sensitivity of MPLQ516E mutationTPO sensitivity test was performed to further investigate the effect of MPLQ516E mutation on the activation of TPOR at different ligand TPO concentrations.MPLWT cells were used as negative controls.Cells in each group were cultured for 96 hours under six different concentration of TPO(2ng/ml,1ng/ml,0.1ng/ml,0.01ng/m,0.001ng/ml,0ng/ml)and then the cell counts in each group was measured by CCK-8assay and statistically analysis was done.9.Activation of JAK2/STAT,PI3K/AKT and MAPK/RAS signal pathway of MPL A497-L498ins4 and MPLW515RQ516E MutantsEach group of Ba/F3 cells were starved without IL3 for 48 hours,and then protein was extracted.Signaling studies were performed on Ba/F3 cell lines by western blot analysis of JAK2(Tyr1007/1008),STAT1(Tyr701),STAT3(Tyr705),STAT5(Tyr 694),p44/p42 MAPK(Erk1/2)(Thr202/Tyr 204),and AKT(Thr 308)and of these different phosphorylated proteins.β-actin is considered as the house keeping protein of all groups.Western blotting was performed by Simple Western Kit(Protein Simple,USA).The activation of JAK/STAT,PI3/AKT and MAPK/RAS signaling pathwaysin each group was analyzed.Results:1.Discovery and identification of two atypical MPL mutations.Through gene mutation screening of MPLexon10 in newly diagnosed triple negative patients,we found two atypical MPL mutations,an insertion mutation MPL A497-L498ins4 and a double point mutation of MPLW515R and Q516E,respectively.We identified the double point mutation by TA-clone and found that the double point mutation of W515R and Q516E was located on the same DNA strand,so we named it MPLW515RQ516E mutation.2.Construction and identification of lentivirus expression vectors of various genes.The CDS of MPL A497-L498ins4,MPLW515RQ516E,MPL W515L and MPL WT weresuccessfullyligatedintothelentivirusexpressionvector pCDH-MCS-T2A-copGFP-MSCV.The gene sequences of each target were identified by Sanger sequencing.The results showed that there were no base mismatch,insertion and deletion in the inserted sequences of the vectors in each group.The MPLQ516E lentivirus expression vector was successfully constructed by site-directed mutagenesis and identified by Sanger sequencing.Each group of lentivirus expression vectors were constructed successfully.3.Cell models of each group was constructedAfter the lentivirus expression vector was successfully constructed,293T cells were co-transfected with three plasmids by liposome method,and the lentivirus particles of MPLA497-L498ins4,MPLW515RQ516E,MPLQ516E,positive control MPL W515L,negative control MPL WT and vector control MSCV were prepared to infect Ba/F3 cell.After 72 hours infection,the efficiency of each group was detected by flow cytometry.The positive clones of the cells with low infection efficiency were sorted to insure the positive rate of GFP in each group was more than 90%.The expression of target gene were identified by PCR and flow cytometry CD110 markers,respectively.All the results showed that the Ba/F3 cell model expressing the target genes stablywas successfully constructed.4.MPLA497-L498ins4 and W515RQ516E mutation promoted the proliferation of Ba/F3without IL-3CCK-8 assay showed that MPL mutants of A497-L498ins4 and W515RQ516E both conferredcytokine-independentgrowthonBa/F3cells.Weobservedthat MPLA497-L498ins4 and W515RQ516E exhibited marked growth on Ba/F3 cells,similar to the W515L,which was used as a positive control,whereas MPLWT,empty vector and un-transfected Ba/F3 cells did not grow optimally.((P<0.001).The cells in MPL A497-L498ins4 group,MPLW515RQ516E group and MPL W515L group proliferated significantly at 48 h and 72 h,which were significantly different from those in the negative control group(P<0.001).However,there was no significant difference in the proliferation ability between atypical MPL mutation(MPL A497-L498ins4 group,MPLW515RQ516E)and MPL W515L group(P>0.05).5.SMPL A497-L498ins4 and MPLW515RQ516E mutations cause hypersensitivity to TPO on Ba/F3 cellsCells expressing MPLA497-L498ins4 and MPLW515RQ516E mutations showed high sensitivity to TPO and grew rapidly compared to MPLWT,empty vector and un-transfected Ba/F3 cells(P<0.001)However,there was no difference between Ba/F3cells carrying MPL mutants and MPLWT at the high TPO concentration(2ng/ml)(P>0.05).6.Cell cycle analysis of MPL A497-L498ins4 and MPLW515RQ516E mutations under different TPO conditions.DNA was labeled by PI,and the proportion of G1,G2 and S phase was detected by flow cytometry.the results showed that the proportion of MPL A497-L498ins4,MPLW515RQ516E and MPL W515L G1 phase decreased in the absence of TPO.61.89%,62.27%,54.68%,respectively,while the proportion of MPLWT G1 phase was 78.63%,which was significantly higher than that of negative control group(P<0.05),MPL A497-L498ins4,MPLW515RQ516E,MPL W515LS phase,30.04%,respectively).33.42%,33.49%,while the proportion of MPLWT S phase was only15.92%,the difference was statistically significant(P<0.05).Under the condition of low concentration of 0.1ng/ml TPO,the proportion of MPL A497-L498ins4,MPLW515RQ516E,MPL W515L and MPLWT was similar to that of G1,G2,S without TPO,and the difference was statistically significant(P<0.05).Under the condition of 10ng/ml TPO,the proportion of MPL A497-L498ins4,MPLW515RQ516E and MPL W515L G1 phase was 65.37%,61.04%and 57.28%respectively,and that of MPLWT G1 phase was 67.67%.There was no significant difference in the proportion of MPL A497-L498ins4,MPLW515RQ516E and MPL W515Ls compared with MPLWT,which were 26.7%,36.92%,34.98%and 26.34%,respectively.7.Effect of MPLQ516E mutation on proliferation of Ba/F3 cells.After withdrawing the IL-3,the single mutation Q516E survived much better than MPLWT(P<0.001),and induced an autonomous proliferation effect on cells but which was weaker than the MPLW515L(P<0.001).Furthermore,the viability of MPLQ516E under various concentration of TPO was also tested.After 96 hours,numbers of cell were identified.Cells stably expressing the mutant Q516E exhibited increased proliferation in response to TPO,in particular at low concentration,when compared to cells expressing MPLWT.8.Activation of JAK2/STAT,PI3K/AKT and MAPK/RAS signaling Pathway in MPL A497-L498ins4 and MPLW515RQ516E Mutants.The phosphorylated proteins referred to JAK2/STAT increased obviously in mutations of MPL(A497-L498ins4,W515RQ516E and W515L).In addition,the phosphorylated AKT and p44/42 MAPK involving in PI3K/AKT and MAPK/RAS pathway were also increased in these two novel MPL mutations.MPLWT,empty vector and un-transfected Ba/F3 cells were used as negative control which activated proteins rarely without cytokine stimulation.Conclusion:These results showed that these two novel MPL mutants can lead to cytokine-independent cell growth,increased TPO sensitivity and G1/S-phase transition on Ba/F3 cells via activating the JAK2/STAT and its downstream pathway.MPLA497-L498ins4 and MPLW515RQ516E mutations are gain-of-function mutations and closely associated with the pathogenesis of TN-MPNs. |
