Construction Of FGFR3Gene Eukaryotic Expressing Vector And Its Functions In Chronic Myeloid Leukemia Cell Line K562

Objective:Constructed eukaryotic expression vector MSCV/puro-fgfr3-WT FGFR3andMSCV/puro-fgfr3-DN, and detected its expression in the human leukemia cell lineK562, gave Imatinib classic treatment of CML while transfected FGFR3-DN,observed the treatment effect of FGFR3-DN transfection preliminary,to lay thefoundation for further studying the role of FGFR3in the treatment of chronic myeloidleukemia and its resistance mechanisms.Method：Obtained full-length FGFR3gene (fgfr3-WT) and truncated inactivated FGFR3(fgfr3-DN) by PCR,connected eukaryotic expression vector MSCV/puro afterdouble digested,constructed MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DNrecombinant plasmids.Transfected them into human leukemia cell line K562byliposome-mediated after PCR, double enzyme digestion and sequencing properly,usedWestern blotting and flow cytometry to detect the expression of FGFR3protein incells after puromycin resistance screening,used CCK8to detect the influence ofproliferation ability of stably transfected K562cells by FGFR3plasmid,used flowcytometry to detect the influence of cell cycle and apoptosis.Used CCK8and flowcytometry method to preliminary detect stably transfected K562cells byFGFR3-DN,and at the same time giving k562cells the drug Imatinib,observed theinfluence of cell proliferation ability.Results：PCR results identified of MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN recombinant plasmid showed that two plasmids were able to amplify the2400bp offgfr3-WT full-length gene fragments and fgfr3-DN truncated fragments of1200bp,indicating the successful amplification of full-length gene fgfr3-WT and1200bp offgfr3-DN gene.Double digested MSCV/puro-fgfr3-WT plasmid also obtained a2400bp fragment of the target gene.Sequence analysis showed that fgfr3-WT size was2400bp.Blast comparative analysis showed that the sequence analysis result wasexactly the same with the FGFR3sequence of GeneBank sequence.Fgfr3-DN sizewas1200bp, fully consistented with the pre-designed sequence,indicating that FGFR3and mutational inactivated FGFR3eukaryotic expression vector plasmid wasconstructed successfully.MSCV/puro-fgfr3-DN transfected group (K562-DN) has ahigher truncated type FGFR3(K562-DN) expression, while the control group and theK562-WT-transfected group did not fragment.Flow cytometry showed that57.5%ofthe fgfr3-WT group cells expressed FGFR3highly and41.5%of the K562-DN groupcells expressed FGFR3-DN highly.CCK8showed that K562cell proliferation wassignificantly increased (p<0.05) compared with empty plasmid group afterFGFR3-DN transfection,and the proliferation of Imatinib-treated group wassignificantly increased (p <0.05) compared with FGFR3-DN-transfected groupalone.Used flow cytometry to detect the cell cycle of FGFR3-DN stably transfectedgroup and combined Imatinib treatment group,the result was that the cell cycle ofFGFR3-DN stably transfected group was longer than empty plasmid group,whileFGFR3-DN-transfected combined with Imatinib treatment group was longer thanImatinib-treated alone group.It indicated that after giving Imatinib,the drug resistancein chronic myeloid leukemia patients may be related to the degree of mutant FGFR3’sexpression and protein function.Specific relationship needs further study. Conclusions：1.Successfully constructed a high expression of wild-type and truncatedinactivating FGFR3expression vector.2.Successfully constructed a high expression of wild-type and truncatedinactivating FGFR3of human leukemia cell line K562.3.Proved FGFR3could promote the proliferation of K562by CCK8and CFUexperiment.