Function Analysis Of Atypical Mutations In Exon13 And 15of JAK2 Gene In Myeloproliferative Neoplasms

Objective:Myeloproliferative neoplasms(MPNs)are a group of clonal disorders characterized by excessive proliferation of one or more differentiated myeloid lineages.At present,studies have fully confirmed that JAK2 V617F,mutations in JAK2 exon12,CALR exon9 and MPL exon10 can independently cause MPNs,suggesting that these mutations can change the protein function of the gene and cause diseases.And the discovery of these mutations can provide a more accurate reference standard for clinical diagnosis and treatment of MPN.However,about 10%-15%of MPN patients do not carry hot spot mutations of the above three genes,which are clinically called"triple negative"MPNs.With the development of Next Generation Sequencing,some atypical mutations outside those hot spots of JAK2 gene have been reported in some"triple negative"patients.However,it is difficult to determine whether they are functional mutations causing MPNs.The exon 13 to 15 of JAK2 encode JH2domain,in which JAK2V617F is also included(exon 14).In our study,we mainly studied whether mutations in exons 13 and 15 of JH2 domain could produce the same functional effect like JAK2V617F.The mutations of JAK2L579F(exon 13)and JAK2 L624P(exon 15)reported in literature were selected for analysis.Our study may have some guiding significance for the functional judgment of the atypical mutations in exon 13 and exon 15 of JAK2 in"triple negative"myeloproliferative neoplasms.Methods:1.Group dividing and construction of lentivirus expression vector of JAK2geneWe have selected the Ba/F3 as the cell model.The experimental groups were designed as:JAK2 L579F group?JAK2 L624P group?JAK2 V617F positive control group?JAK2 WT group?JAK2 MSCV group and Ba/F3 cell group.JAK2 L579F and JAK2 L624P were directly obtained from DNA synthesis technology.And JAK2 WT and JAK2 V617F were obtained by RT-PCR.Later we amplified them by PCR,then the recombinant expression vector pCDH1-MSCV-MCS1-T2A-copGFP was constructed with those by restriction enzyme digestion and ligase connection.2.Construction of the Ba/F3 cell models stably expressing JAK2 L579F?JAK2 L624P and other control group293T cells were cotransfected with the constructed expression plasmids and the packaging plasmids psPAX2?pMDX1 respectively in a certain proportion by lipo2000 liposome(the cell line passages are required to be within 15generations).After 48 hours of transfection,lentivirus suspension was collected.Then filtered it with 0.45uM filter,collected with 5ml EP tubes.And stored in refrigerator at-80?.Meanwhile,293T cells were placed under laser confocal microscope to observe the expression and distribution of GFP.Take the sterile24 well plate,add Polybrene(final concentration 6?g/ml),400?l Ba/F3 cell culture medium,Ba/F3 cell(2*10~5),1.6 ml lentivirus suspension into the 24well plate under the ultra clean bench.Mix gently,culture in 37??5%CO2incubator for 24h,and then replace the fresh medium with 15%FBS.After 48 to72 hours,GFP expression of Ba/F3 cells was observed by laser confocal microscopy.After that,the GFP expression ratio was measured by FACS.And then expand culture of infected cell lines.RT-PCR and Western bolt were used to detect the expression of the target genes in the clone,and the stable cell lines would be established and preserved.3.Effect of JAK2 L579F and JAK2 L624P on the proliferation of Ba/F3Detect the proliferation of cells in each group by CCK8.Ba/F3 cells must rely on IL-3 to grow.So in our study,by withdrawing IL-3,the absorbance of each group of cells at different time points(0-5d)was measured to reflect the cell proliferation.Each group of cells was set up with three multiple pores,and this experiment was repeated for three times.4.Analysis of G1?G2?S-phase of cell cycles of JAK2 L579F and JAK2L624P mutations without IL-3DNA of these cells was labeled with propidium iodide(PI).And the G1?G2and S phase of cells in each group were detected by flow cytometry.In our study,the cells(1*10~6)of each group were cultured 48h removing from IL-3.After cleaning the residual culture medium with sterile PBS,70%ethanol was used to fix the cells overnight at 4?.Next day,after incubation with PI for 30 minutes,the proportion of G1?G2 and S phases of cell cycle in each group was detected by flow cytometry.5.The role of JAK2 L579F and JAK2 L624P in JAK2/STAT signal transduction pathwayJAK2 mutation groups and other control groups were washed with PBS for three times.And each group of cells was resuspended in 1640 complete medium without cytokine according to 4*10~4/ml.After 48 hours of culturing,the cells were collected and the total protein was extracted.BCA method was used to quantify the protein.Western blots was used to detect and analyze the phosphorylation protein related to JAK2/STAT signal pathway in each group of cells.6.Construction of crystal protein and molecular dynamics simulationThe protein sequence of JAK2 gene(UniProt:p060067)was obtained,and the crystal containing L579 and L624 was selected to label the mutation sites.The mapping tool PDBSWS was used for site mapping,and PyMOL was used for drawing.The sander module of amber18 was used to complete the molecular dynamics simulation.The dynamic simulation was carried out including energy minimization,heating,equilibrium and production running.The trajectory was completed by CPPTRAJ module.MMGBSA in amber was used for calculating the binding affinity.Results:1.Construction and identification of Lentivirus Expression VectorsEach gene,named JAK2 L579F?JAK2 L624P?JAK2 V617F?JAK2 WT and JAK2 MSCV,was successfully constructed into the lentivirus recombinant expression vector pCDH1-MSCV-MCS1-T2A-copGFP.And the bands were in corresponding positions by horizontal gel electrophoresis.At the same time,after PCR amplification,Sanger sequencing,and comparison with JAK2 gene in NCBI database,there was no mismatch,loss or increase of bases.2.Construction and identification of stable cell modelsThe virus suspension of lentivirus recombinant expression vectors were collected and infected with Ba/F3 cells.After 48 hours,green fluorescence was observed in the cells of each group by fluorescence confocal microscopy.And after 72 hours,flow cytometry was used to detect the cell infection efficiency of each group.The GFP expression efficiency of each group was more than 90%.PCR was used to identify the expression of target mRNA in each group.3.Analysis and comparison of the proliferation ability of Ba/F3 cell model in each groupCCK-8 was used to detect the growth of Ba/F3 cells independently of IL-3at different times in each group.The results showed that there was no significant cell proliferation between JAK2 WT?JAK2 MSCV and Ba/F3 groups at each time.What's more,there was no statistical difference between each group(P>0.05);while JAK2 L579F?JAK2 L624P and JAK2 V617F groups had significant proliferation during 48h and 72h,compared JAK2 WT?JAK2 MSCV and Ba/F3 groups(P<0.001).But the cell proliferation ability of JAK2 L579F and JAK2 L624P were also significantly different from that of JAK2 V617F positive control group(P<0.05),suggesting that there was only weak proliferation effect in JAK2 L579F group and JAK2 L624P group.4.Cell cycle analysis of Ba/F3 cell models without IL-3PI was used to label DNA in cells.And flow cytometry was used to detect the proportion of different stages in cell division interval.In JAK2 WT group,the proportion of G1 phase was 95.05%,and that of S phase was 94.8%.Without IL-3,the ratio of G1 phase in JAK2 L579F?JAK2 L624P and JAK2 V617F were decreased compared with JAK2 WT,which were 73.65%,78.7%and67.49%,respectively.However,S-phase increased,23.93%,20.93%and 30.79%respectively.There was no significant difference between the JAK2 L579F?JAK2 L624P and JAK2V617F positive control group(P>0.05).But there was significant difference between the two experimental groups and JAK2 WT negative control group(P<0.05).5.Activation of JAK2 L579F and JAK2 L624P in JAK2/STAT signaling pathwayThe expression levels of P-JAK2,P-STAT3 and P-STAT5 in JAK2 L579F and JAK2 L624P groups were significantly increased.There was no significant difference between JAK2 V617F group.However,in JAK2 WT group?JAK2MSCV group and Ba/F3 group,there were a litter or none expression levels of phosphorylation proteins of JAK2/STAT.6.JAK2 L579F and JAK2 L624P could interfere with the binding affinity between JAK2 and its substrate ATPThe crystal structure showed that JAK2 L579F and L624P could change the spatial structure of JAK2 JH2,and the binding site of JAK2 JH2 with ATP would also be changed greatly.The results of molecular dynamics simulation showed that the binding affinities of the two mutations were more than twice than that of the wild type,which implied they could greatly interfere with the binding ability of their substrate ATP,and then change the function of the protein.Conclusion:1.In this study,JAK2 L579F and JAK2 L624P mutations were constructed into cell models,and confirmed that mutations in the 13 and 15 regions of JAK2exon can produce the same cell proliferation effect as JAK2V617F.These results suggested that mutations in exon 13 and 15 of JAK2 may play a driving role in myeloproliferative neoplasms.Although the mutations in this region can be considered as a functional mutation in clinical analysis,we still need to exclude some unaffected mutations.2.Crystal structure constructing and Molecular dynamics simulation demonstrated that L579F and L624P could change the spatial conformation of JAK2 protein,but these two mutations cause JH2 instability by interfering with the binding ability of JAK2 protein and its substrate ATP.However,JAK2V617F could change the stability of JH2 by reducing the TM value of?C structure of JH2 domain.But the final effect was to cause the inhibition effect of JH2 domain on JH1 domain for all of them.