Experimental Study On The Effect Of Human NRAGE On Cell Growth And Cell Migration

Neurotrophin receptor p75 interacting MAGE homologue (NRAGE, MAGE-D1), belongs to the MAGE (melanoma antigen) protein family and contains 13 exons, 11 of which encode a protein of 86kDa. hNRAGE, the human homologue of NRAGE was cloned from a human placenta cDNA library, which can inhibit proliferation of hepatocellular carcinoma cells when it is overexpressed.Until now, NRAGE has been shown to be involved in cell cycle arrest and apoptosis regulation in several studies, but the normal physiological function of NRAGE is unknown.In this paper, we employed a recombinant adenovirus to research the effect of human NRAGE on cell growth and cell migration both in vitro and in vivo. Main results are as follows.First, by the way of homologous recombination, we constructed a recombinant adenovirus that could express human NRAGE effectively in eukaryotic cells and testified the function of the adenovirus through immunoblotting.Second, FACS analysis showed that HEK 293, U2 OS and B16-BL6 cells which overexpressed human NRAGE by recombinant adenovirus or eukaryotic expression plasmids were arrested at G1/G0 or G2/M phase while the percentage of cells at S phase was reduced. Moreover, B16-BL6 cells infected with Ad-myc-hNRAGE developed melanoma much more slowly in C57BL/6J mice in contrast with control groups.Third, in vitro migration assay including wound-healing and transwell experiments indicated that migrating speed of B16-BL6 cells infected with Ad-myc-hNRAGE was inhibited. When B16-BL6 cells were injected through the tail vein of C57BL/6J mice, B16-BL6 cells overexpressed NRAGE formed much less...