Expression Of NDRG2 Gene In Breast Cancer Tissue And Its Effect On Proliferation Of Breast Cancer Cell

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 expression of NDRG2 gene in 80 fresh breast cancer tissue collections and corresponding tissue around breast cancer (including infitrating ductal carcinoma, n = 54; lobular carcinoma, n = 18; lobular carcinoma in situ, n =8) by immunohistochemistry, real time PCR, western blot and so on. Results indicate that mRNA expression of NDRG2 is lower in 63 breast cancer tissue collections compared with corresponding tissue around breast cancer, and is positive correlation to tumor grade; higher in 5 breast cancer tissue collections compared with corresponding tissue around breast cancer. In addition, there is not significant different in mRNA express of NDRG2 between 12 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.2. To explore the effect of gene NDRG2 transfection on the proliferation in breast cancer cell. By the induction of liposome , pcDNA3.1- NDRG2 and eukaryotic expression vectors pcDNA3.1 ( +) were transfected into breast cancer cell line SK-BR-3. Then the subclone cell lines SK-BR-3- NDRG2 which expressed NDRG2 stably and higher were obtained by persistent G418 selection.. Which establish a solid ground for the subsequent in vivo and in vitro experiments. In order to observe the effect of NDRG2 gene expression on Bcap37 cell line,which expresses low level NDRG2 gene is transfected with recombinating adenovirus expressing high level NDRG2 gene to improve NDRG2 gene express level.3. The proliferation activity of the cell lines was detected by plate colony formation,MTT and flow cytometry. The results indicate that high expression level of NDRG2 gene inhibits cells growth and arrest cells in G1 phase ,which suggest that NDRG2 may inhibit cell proliferation by cell cycle arrest.To sum up, the express of NDRG2 gene in breast cancer tissue increases with breast cancer grade. Overexpression of NDRG2 in SK-BR-3 cells and Bcap37cells effectively inhibited cell proliferation and induced cell cycle arrest. 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.