Cloning And Expressing Of Human GRIM19 And Investigating It's Anti-Proliferation Activity

Signal transducer and activator of transcription 3(Stat3) is a latent(cytoplasmic) transcription factor that can be activated by cytokinesand growth factors.Stat3 plays important roles in cell growth,anti-apoptosis and cell transformation, and is constitutively active invarious human cancers. GRIM-19 ( Genes associated withRetinoid-IFN-induced Mortality ) is identified as a novel celldeath-regulatory molecule,represses Stat3 transcriptional activity and itstarget gene expression, and also suppresses cell growth inSrc-transformed cells and a Stat3-expressing celline.Carrier is required fortargeting the drug to tumor. A 31-aa peptide (F3) that encodes anN-terminal fragment of human high mobility group protein 2 (HMGN2),can carry a payload (phage, fluorescein) to a tumor and into the cell nucleiof the cancer. This peptide may be suitable for targeting cytotoxic drugsand gene therapy vectors into tumors. In this work,we cloned GRIM19 cDNA and constructed F3-GRIM19express vector.Then we expressed the protein in prokaryote cells and testedit's anti-proliferation biological activity for further research of the structureand function of GRIM19 and to pave a way to cure human tumors. First, total RNA was extracted from Chinese placenta tissue. TheGRIM19 cDNA was amplified from total RNA by RT-PCR, then clonedinto pET32a(+) and sequenced. Thus, the prokaryotic expression vectorspQE30-GRIM19 and pQE30-F3-GRIM19 were constructed by subclone,and the fusion proteins was expressed after IPTG induction. We purifiedthe fusion proteins inclusion body by using Ni2+-NTA affinitychromatography ,and renatured them by density gradual dialysis. Second,we investigated the fusion proteins' anti-proliferation to nomalcells and tumor cells by MTT analysis.Then we constructed the eukaryoticvector pcDNA3.1-GRIM19 by subcloning, then transfected it into tumorcells and nomal cells and let them express transiently, tested the effect ofGRIM19' gene expression on the proliferation of tumor cells and nomalcells. In conclusion,we have cloned human GRIM19 cDNA andconstructed pQE30-GRIM19 and pQE30-F3-GRIM19 prokaryoticexpression vectors successfully . The fusion proteins expressed inEscherichia coli SG13009 were insoluble inclusion body, the fusionproteins accounted for up to 56% of total protein in the transformedbacteria. Soluble fusion protein was obtained by Ni2+-NTA affinitychromatography and renaturing. The molecular weight were about 18kDaand 25kDa. The purity of the product was up to 90 percent. The biologicalactivity was measured by MTT, the F3-GRIM19 protein can supress tumorcell' growth. The transient expression of GRIM19 gene bypcDNA3.1-GRIM19 can inhibit cancer cells proliferation in vitro,whilefew effect on nomal cell.The successful expression of human GRIM19 laidthe foundation for the anti-tumor therapy.