| The NDRG2 gene(N-myc downstream regulated gene 2) was first discovered and cloned from normal human whole brain cDNA library in 1999. This gene locates on human chromosome 14q11.2,including 16 exons and 15 introns, and its full mRNA is 2,024bp in length, encoding the 357 amino acid,40KD NDRG2 protein. NDRG1 was the first member found of NDRG gene family, which was isolated in N-myc mutant mouse embryos and found be repressed by N-myc and c-myc. Human NDRG gene family consists of 4 members: NDRG1, NDRG2, NDRG3, NDRG4, which has high similarity in nuclear acid or amino acid sequence. However, their expression levels in spatio-temporal distribution and periods of cells and tissues development are distinct.The knowledge about the function of the NDRG gene family came mainly from the studies on NDRG1. Now, many intracellular and extracellular factors have been found which could regulate the expression of NDRG1. Homocystein, p53, retinoic acid, oxidative and stress condition such as hypoxia, nickel and other reductive agents, et al, can upregulate NDRG1 expression. Inferring from all the references obtained, the functions of NDRG gene family may be involved in cellular differentiation events, maintaining the balance of cell redox potential, and counteracting cell malignant transformation and so on.Our preliminary studies showed that the mRNA expression patterns of NDRG2 gene was dominant in the white matter, gray matter and neuron of brain, salivary gland, skeletal muscle, and lower in bone marrow, testis, peripherial blood and placenta. However NDRG2 gene did not express in leukocyte, colorectal cancer and some tumor cell line such as Hela S3, leukemia cell, Burkitt's lymphoma, adenocarcinoma SW-480, all above suggested that NDRG2 gene had the relationship with tumor, might be a new suppressor gene candidate. So, to study its function and regulation has important theoretical meaning and foreseeing clinical value, but up to now, there have been no reports about the precise cellular and molecular function of NDRG2 gene. Our research is aimed to observe the effect of NDRG2 gene on tumorigenesis of breast cancer, and explore its preliminary function. The main finding of this research include:1. Detecting mRNA and protein express of NDRG2 gene in human breast cancer tissue and breast cancer cell lines by immunohistochemistry, real time PCR, western blot and so on. Results indicate that the express level of NDRG2 in breast cancer cell lines MCF-7, MDA-MB-231 and SK-BR-3 is significantly lower than that of GES; but there is not significant different in the express level of NDRG2 between breast cancer cell lines Bcap-37 and GES.2. NDRG2 is detected in 40 fresh breast cancer tissue collections and corresponding tissue around breast cancer (including infitrating ductal carcinoma, n = 20; lobular carcinoma, n = 12; lobular carcinoma in situ, n = 8) by the above methods. Results indicate that mRNA express of NDRG2 is lower in 23 breast cancer tissue collections compared with corresponding tissue around breast cancer, and is positive correlation to tumor grade; higher in 4 breast cancer tissue collections compared with corresponding tissue around breast cancer. In addition, there is not significant different in mRNA express of NDRG2 between 13 breast cancer tissue collections and corresponding tissue around breast cancer. So the results suggest that the express of NDRG2 gene is negative correlation to reproductive activity of cancer cells.3. To investigate the possible reasons of low express of NGRG2 gene in 3 different breast cancer cell lines by multiplex genomic PCR and methylation sensitive restricted cleavage enzyme analysis. Results indicate that deletions and loss of heterozygosity may cause the low express of NGRG2 gene in MCF-7 cell line, while DNA methylation in upstream promoter may be the reason of low express of NGRG2 gene in SKBR-3 cell line. However in other breast cancer cell lines such as MDAMB-231 cell line, DNA methylation in upstream promote and deletions and loss of heterozygosity are not found, which suggest that some other factors might inhibit the express of NDRG2 gene.4. pAdTrack-CMV adenovira expression system (Novagen corporation, American) is successfully applied to get high titer recombinating adenovirus (1.1×1011pfu /ml) expressing high level NDRG2 gene. Which establish a solid ground for the subsequent in vivo and in vitro experiments.5. In order to observe the effect of NDRG2 gene express on MCF-7 cell line, MCF-7 cell line expressing low level NDRG2 gene is transfected with recombinating adenovirus expressing high level NDRG2 gene to improve NDRG2 gene express level. Cell shrinkage and cell membrane scrambling are observed in some cells by using phase-contrast microscope after 48 hours transfecting pAd-NDRG2, while no marked changes in cell shape in MCF7 cells tranfecting pAd-plasmid vector. MTT results indicate that NDRG2 express inhibits MCF7 cells growth. Flow cytometry results indicate that high express level of NDRG2 gene results in G1 phase arrest in MCF7 cell line, which suggest that NDRG2 may inhibit cell proliferation by cell cycle arrest. Apoptosis experiments indicate that compared with non-transfected MCF7 cells and MCF7 cells transfected by empty vector, apoptosis cells obviously increase in MCF7 cell line transfected by recombinating adenovirus expressing high level NDRG2 gene, 12% after 48 hours and 21.5% after 72 hours, which is identical with DNA Ladder. These results suggest that over-expressed NDRG2 gene may cause apoptosis of MCF7 cells.6. Establishing transplantation tumor model in athymic mouse by inoculating MCF7, pAd-MCF7 and pAd-NDRG2-MCF7 subcutaneouly in mouse back. Results indicate that compared with athymic mouse inoculating MCF7 cells and athymic mouse inoculating pAd-MCF7 cells, tumors grow slowly and volume and weight of tumors decrease significantly in athymic mouse inoculating pAd-NDRG2-MCF7 cells. And immunohistochemistry shows that NDRG2 express up-regulate obviously, while NDRG2 express level is low in athymic mouse inoculating MCF7 cells and athymic mouse inoculating pAd-MCF7 cells. These results suggest that NDRG2 medicated by adenovirus can persist expressing and inhibit cancer cells development.To sum up, the express of NDRG2 gene in breast cancer tissue and breast cancer cell lines increases with breast cancer grade, and the reason of low express of NDRG2 gene might be related to deletions and loss of heterozygosity and DNA methylation in upstream promoter. In athymic mouse inoculating pAd-MCF7 cells model, over-express of NDRG2 gene significantly decrease tumors grow as well as volume and weight of tumors. These findings about the different expression and function of NDRG2 gene in breast cancer tissue and breast cancer cell lines is helpful to confirm the function of NDRG2 gene, which will provide theoretical base for mechanisms of breast cancer and gene therapy for breast cancer. |
