Clone Of Pro-oncogene Pim-3 CDNA And Its Effect On Apoptosis Of Hepatoma Carcinoma Cyte

AIM: To clone rat pim-3 cDNA and construct its pim-3 eukaryotic expressing plasmid and to investigate the expression of pim-3 in eukaryocyte and its effect on apoptosis.METHODS: Total RNA was isolated from rat skeletal muscle tissues with TRIzol.pim-3 cDNA was made by RT-PCR and constructed into eukaryotic expression vector pEGFP-N₂, which expresses green fluorescent protein.The recombinant pEGFP-N₂/pim-3 was then transformed into E.coli JM109 host bacteria.After authenticated by restriction digestive enzymes and sequencing, plasmid pEGFP-N₂/pim-3 was transfected into SMMC-7721 cells by liposome. Fluorescent microscope was used to examine the expression of pim-3 , and the biologic behavior of transfected cells was tested by flow cytometry and MTT.RESULTS: Restriction enzyme digestion analysis and sequencing showed that the recombinant plasmid of rat pim-3 was correctly constructed; Flow cytometry showed the percentage of apoptosis cells transfected by the recombinant plasmid pEGFP-N₂/pim-3 was 3.48%,and the plasmid pEGFP-N₂ transfected cell group was 10.74%(P<0.05), and non-transfected cell group was 11.01%,there were signicant differences between the recombinant plasmid transfected group and the other two control groups(P<0.05); Fluorescent microscope and MTT showed a pim-3 expression in SMMC-7721 cell and the apoptosis of the transfected cells was significantly inhibited.CONCLUSION: Our study showed that pim-3 gene was successfully cloned .It may be feasible to use as an inhibitor of apoptosis.