Research Of Cellular Expression And Localization Of Ndrg2

Background:The human NDRG2 (N-myc downstream regulated gene 2) gene was first discovered and cloned from normal human whole brain cDNA library in our lab in 1999. This gene locates on human chromosome 14q11.2, including 16 exons and 15 introns, and its full mRNA is 2,024 nt in length, it is identified as a new gene by BLAST, which encodes a 357 amino acids, 40 kD Ndrg2 protein. The study indicates that this gene expresses in many tissues, and is a different expression gene between normal brain tissue and glioma. It is reported that NDRG gene family consists of 4 members: NDRG1, NDRG2, NDRG3 and NDRG4, and NDRG4 can be divided into three subtype: NDRG4-B, NDRG4-Bvar和NDRG4-H。These four members have high similarity in amino acid sequences, but their expression levels are quite different in many tissues. Most of the studys focus on NDRG1 and NDRG2. The datas show that many factors can up-regulate NDRG1 such as metallic compound(Ni2+, Co2+), hypoxia, DNA damage, vitaminD and retinoids, androgen and P53; whereas NDRG1 is down-regulated by protein Myc and protein VHL, and is also down-regulated in tumors. It has been reported that NDRG1 plays an important role in the process of cell proliferration and differentiation, synthesis and metabolism of lipid, responses of cell stress, generation and matastasis of tumors,and also it is reported that NDRG2 involves in the process of cell differentiation, cell stress, generation and matastasis of tumors. Someone finds that when cells are in the circumstance of hypoxia, the Ndrg1 protein translocates from the cytoplasm to the nucleus. Because Ndrg2 and Ndrg1 have high homology, and both of them are found to be involved in the process of cell differentiation, cell stress, generation and matastasis of tumors, we assume that this two genes are similar on the part of functions to some extent. Then we studied cellular localization of endogenous Ndrg2 protein on cell stress, the results show that Ndrg2 protein translocate from the cytoplasm to the nucleus under some kinds of sreesses. There is no classical NLS in Ndrg2 by the analysis of bioinformatics. So, Ndrg2 may be transported into the nucleus by other known or unknown ways. The research on the identification of the phenomena of Ndrg2 transported into the nucleus under conditions of sresses and the possible mechanism of this transportation can partly clarify the possible functions of Ndrg2, and provides clues for the future research.Purpose and content: Identify the expression and cellular localization of Ndrg2 in many cell lines; detect the cellular localization of Ndrg2 in many cell lines in the conditions of stress; set up the cell model of Ndrg2 transportation from the cytoplasm to the nucleus in the circumstance of stress; explore the possible mechanism of Ndrg2 translocation from the cytoplasm to the nucleus by transfection of the truncated NDRG2 into cells.Methods:1. Collecting 31 kinds of human cell lines, detect the endogenous expression of Ndrg2 by Western-blot, select the cell lines with high expression of Ndrg2 and suitable cell shape, then detect the sub-cellular localization of Ndrg2 in normal conditions by indirect immunofluorescent.2. Stimulate the cells with many kinds of stress, then on one hand isolate the cytoplasm part and the nucleus part of cells and carry out Western-blot to detect weather Ndrg2 translocate from the cytoplasm into the nucleus in the conditions of stress; on the other hand identify the sub-cellular localization of Ndrg2 in the conditions of stress by indirect immunofluorescent.3. Transfect the truncated NDRG2 into cells, observe the cellular localization of truncated NDRG2 in the conditions of stress.Results:1. The expression of Ndrg2 is identified in 31 cell lines; and Ndrg2 localize in the cytoplasm of the cell lines that are detected in normal conditions; we also find that in the cell lines that are detected the Ndrg2 protein may colocalize with mitochondria in normal circumstance.2. A cell model of translocation of Ndrg2 from the cytoplasm into the nucleus in the conditions of hypoxia stress is established: the condition can be described as follows; that the A549 cell line or the Bcap37 cell line is treated with 0.5mM or 1.0 mM NiCl2 for 12 hours.3. Successfully build a recombinant human eukaryotic expression vector of NDRG2.4. Validate the translocation of Ndrg2 from the cytoplasm to the nucleus in the conditions of stress by transfection of the truncated NDRG2.5. We find a continuous existing band in the experiment of Western-blot, and this protein band exists in the part of the nucleus of every sample that is detected, and this protein band is identified as a possible product of a new splicing variant of NDRG2 by bioinformatics and records research.We successfully build the EGFP fusion expression vector of the possible splicing variant of NDRG2, and find the expressed fusion protein continuous existing in the nucleus and the distribution of the fusion protein in the nucleus is mottled.Conclusion: The Ndrg2 protein localize in the cytoplasm of many cell lines; in normal conditions, the Ndrg2 protein may colocalize with mitochondria; both of endogenous and ectogenesis Ndrg2 can translocate from the cytoplasm to the nucleus in the conditions of hyposia stress; a new splicing variant of NDRG2 may exist.