Advances In Subcellular Localization Of NRAGE Protein And Its Function

Background&Objective:NRAGE (neurotrophin receptor p75-interacting MAGE homolog) gene is melanoma associated antigen MAGE family member which involved in apoptosis, cell cycle regulation and tumorigenesis process. The first part of preliminary experiment shows that protein NRAGE in HELA cells has high distribution proportion in nucleus, but recent studies about NRAGE is based on it as a cytoplasmic protein, so the subject designs experiments to disscuss the following questions:1. Analyzing the different distrubution of protein NRAGE between normal cells and cancer cells;2. Analyzing nuclear localization mechanism of protein NRAGE;3Analyzing the nuclear isoform function.Methods:Detecting protein NRAGE distrubution in normal cells and cancer cells by using immune fluorescence; Analyzing protein NRAGE nuclear localization mechanism by constructing subclone of NRAGE isoforms; Analyzing NRAGE nuclear isoform function by hydrogen peroxide stimulation experiment.Detecting protein LC3B expressiong after D-HANKS Solution, m IFN-y, chloroquine stimulation to analysis whether protein NRAGE is involved in autophagy; beta-galactosidase detects senescence degree of NRAGE gene wild-type and knockout mouse fibroblasts; Q-PCR detects inflammatory cytokine expression in NRAGE gene wild-type and knockout mouse peritoneal macrophages after LPS stimulation.Results:Protein NRAGE in normal such as HEK-293cells, C2C12cells, HSF cells, MSC cells are mainly localized in the cytoplasm, and in cancer cells such as HELA cells, HepG2cells, MDA-MB-231cells, MCF-7cells proteins NRAGE has high distribution proportion in nucleus; plasmid of NRAGE isoforms (920aa,834aa,324aa subtypes) are localized in the cytoplasm; HELA cells has an increased expression of NRAGE after hydrogen peroxide stimulation.LC3B â…¡ in NRAGE gene knockout mouse fibroblasts has an increased expression than the wild type in control group and stimulation group; we subculture the embryonic fibroblasts to generation sixth,senescence degree of NRAGE gene knockout mouse fibroblasts is higher than wild type; NRAGE gene knockout mouse peritoneal macrophages have a higher susceptibility to inflammatory stimuli. Conclusion:Mechanism of protein NRAGE nuclear localization may be gene alternative splicing, and protein NRAGE nuclear isoform may involve in DNA damage repair process, further hypothesizing that NRAGE nuclear protein isoform may have relevance with Tumor.Protein NRAGE involved in lysosomal autophagy process, it may involve in an autophagy induced senescence process, this NRAGE involved process may be associated with inflammation.