Primary Study Of The Role Of NDRG2 In Tumor Cells

Human NDRG2(N-myc downstream regulated gene 2) was first discovered by FMMU of China, which was registrated in Genbank with No. AFI59092. NDRG2 is expressed widely in normal tissues and differently between normal and tumor tissues through in-site hybridization. But the mechanism and process of the gene in regulation on cells is still not clear.In this study, the NDRG2 gene was cloned into PQE30/M15, and DNA Sequencing proved the construction of the plasmids was correct. After expressed by the IPTG induction, MW of the proteins expressed by the vector is correct. We refold the protein through quick dilution.After desalt,we purified the protein by the affinity resin.The isoelectric point (PI) of the protein was 6.3.N-terminal protein sequencing proved the protein N- terminal amino acid construction was correct.We investigated inhibitory activity of the NDRG2 protein to the HHCC and 7901 cells by setting up the different protein concentration and control groups. The cell cycle were analysed by two-parameter flow cytometry. The in vivo effects were evaluated by tumor -inhibition test on nude mice. These results indicated that the recombinant NDRG2 proteins had some inhibited effects on tumor cells, causing G1 phase arrested and inhibiting tumor formation on nude mice in vivo.Also we have constructed a plasmid containg the shRNA of NDRG2 to suppress the expression of NDRG2 in HHCC cell, and a plasmid containing NDRG2 to improve the expression of NDRG2. Our purpose was to test the cells properties of improving or reducing the NDRG2 content. A 336 bp human U6 snRNA promoter was amplified from human genomic DNA by PCR and ligased with a 29 bp reverse repeated motif of NDRG2 targetsequence with 9 bp spacer and plasmid pSNAV. The recombinant pSNAVPU6 plasmid transfected with HHCC cell lines to detect the effects of NDRG2 expression separately. The analysed results indicated that pSNAVPU6 suppressed the NDRG2 expression successfully and caused Gl phase arrested. The transfected cells had less ability of colony formation and tumor formation on nude mice in vivo, than the control ones. The above results implicates that there may be a kind of nuclear translocation sequence in NDRG2.Finally, we did some genechip analysis about cell apoptosis and human cancer pathwayfinder on the cells of adding and reducing NDRG2 expression. The results indicated that there was no direct relationships between expression level of NDRG2 and cell apoptosis. However, NDRG2 had some effects on the cancer pathwayfinder genes. NDRG2 has negative control on p21.lt may stimulate growth of cancer cells by upregulating vascular endothelial growth factor receptor Tie-2. Similary, it has negative control on CD40L. We concluded that NDRG2 put up some characters of cancer genes in some cells. It need more tests to make it clear about the mechanism.