Study On Potentiality Of Malignant Transformation And Drug-Sensitivity Of The PDGFRA Mutation In Gastrointestinal Stromal Tumor

Aims:By observing the action of the L839P which is a new site of the PDGFRA mutation in the gastrointestinal stromal tumors to the Chinese hamster ovary cell about the cell proliferation,apoptosis,expression of protein,the neoplasia in the nude mouse and the sensitivity to imatinib to identify the action of malignant transformation of PDGFRA gene mutation in the development of the gastrointestinal stromal tumors and the influence to the imatinib-sensitivity of such tumors.Methods:1.Based on the previous studies of laboratory,collecting 25 new cases of fresh GISTs in the gastrointestinal tract,mesentery and omentum,extracting genomic DNA,combining the PCR amplication and direct sequencing to identify the exon12 and exon18 mutations of PDGFRA to screen the new mutant sites.2.Stably transfecting the recombinant plasmids pcDNA3.1 empty vector (NP),PDGFRA wild-type(W),PDGFRA L839P(M1),PDGFRA D842V(M2) respectively into the CHO cells by liposome methods and detecting the expression of PDGFRA protein by Western-blotting;describing the cell growth curve by cell counting,discovering the cell cycle with flow cytometry,detecting the cell apoptosis with Annexin V,to make it clear that the actions of the PDGFRA gene missense mutation L839P to the cell proliferation and apoptosis.3.Subcutaneously injecting the stably transfecting cells into the back of the nude mice to observe the tumorigenesis.4.Transient transfecting the mutant(M1,M2) plasmids of PDGFRA respectively together with the wild-type(W) plasmids of KIT into the CHO cells,detecting the expression of KIT protein and phosphorylated forms of which with Western-blot methods to reveal the impacts of the PDGFRA mutants towards the KIT wild-type about the protein's expression and phosphorylation while coexist.5.Co-incubating the four stably transfecting cells respectively with the imatinib which are various concentrations for seventy-two hours respectively to detect the cell proliferating activity;After transfecting the mutant and wild-type plasmids of PDGFRA and KIT respectively into the CHO cells for twenty-four hours,incubating which with imatinib with different concentrations for ninty minutes,then extracting the cell lysates to detect the protein expression and phosphorylated status,by which to investigate the influence of the mutants to the imatinib-sensitivity of the GIST.Results:1.We identified one substitute mutation Y849H in the exon18 of the PDGFRA in 25 cases,which lead to the substitution of Tyr to His.There is no mutation be identified in the exon12.2.The lysates from four stably transfecting cells displayed the expression of PDGFRA protein in the wild-type(negative control)and the mutant(experimental group and positive control) except the empty vector.The growth curve displayed the growth velocity speed up in the experimental group and positive control.The cell cycle detection presented the ratio of cells in proliferative phase(S+G2-M) were 28.38%(blank),24.47%(negative control),43.79%(experimental group) and 40.89%(positive control).The detection of cell apoptosis show the apoptotic indexes were 1.77%,1.90%,1.45%,1.57%in turns.3.After three weeks,the neoplasms were observed in the nude mice which injected the stably transfecting cells of experimental group and positive control.The microscopic presentation showed the epithelioid cell morphology.4.The expression of the phosphorylated protein of KIT strengthened while co-transfecting the mutant plasmids of PDGFRA and the wild-type plasmid of KIT.5.After seventy-two hours,compared with the positive control,blank and negative control,the experimental group showed obvious proliferated inhibition while the drug was higher concentration.After transient transfection and co-incubation shortly,the lysates showed that the expression of phosphorylated protein of M1,S and Po suppressed obviously.Conclusions:1.The point mutation Y849H in the exon18 of PDGFRA gene was detected in this subject located in the hot site reported in the reference,but the type of mutation was different.The incidence of mutation was 4%.This case occurred in the stomach,the largest diameter was 2.2 centimeters and the biological behavior was low potential malignancy.2.Detecting the expression of the plasmids in the cells,it confirmed that each recombinant plasmid had transfected stably into the CHO cells and expressed the products. 3.Compared with the blank and the negative control,the mutants could make the growth velocity speed up and reinforce the proliferative activity,make the proportion of the cells which is in S+G2-M higher and the index of apoptosis lower.It presented that this PDGFRA mutation L839P is gain-of-function mutation.4.Succeeding to induce the tumorigenesis in the nude mice showed that the PDGFRA mutation L839P had more malignant transformation effects to the cells.It suggested that the PDGFRA mutation is one of the most important mechanisms in the generation of GIST.5.PDGFRA mutation could activate the wild-type KIT protein and make it autophosphorylate without the present of ligand.It suggested that except for activating the PDGFRA protein,the PDGFRA mutation could make the KIT protein activated and induce the downstream signal transduction cascades,which could promote proliferation and inhibit apoptosis,lead to the tumorigenesis at last.6.The GIST contained either PDGFRA L839P or KIT Ins IPYD579,Del 559-560 mutations reacted to the imatinib therapy,but the PDGFRA D842V was not sensitive.