Temporal And Spatial Distribution Of NDRG2 Expression In Human And Mouse Tissues And Preliminary Study On Its Function

[BACKGROUND] N-myc downstream regulated gene2 (NDRG2), together with NDRG1, NDRG3 and NDRG4, constitute the new NDRG family. Although NDRG family is believed to be involved in cell growth, the exact molecular and cellular function of members of this family is still unknown. Human and mouse NDRG2 that share 95% amino acid sequence identity were discovered and successfully cloned by our research group and another group respectively. NDRG2 are located at chromosome 14q11.2, including 16 exons and 15 introns, and its cDNA is 2024 base pair (bp) in length. Studies from our laboratory and from others have shown that NDRG2 was highly expressed in many normal tissues, whereas lowly expressed in various tumor tissues and tumor cell lines, therefore, suggesting that NDRG2 may be play a important role in some tumor genesis and evolution. A recent study showed that NDRG2 regulation may be associated with disease pathogenesis in the brain, such as Depression and Alzheimer's disease (AD). However, the functional role of this gene is still unclear under some pathological and physiological conditions. [AIM] 1. To make clear the trend and cellular location of NDRG2 expression in mammal development, the distribution characteristic of NDRG2 expression was systematically analyzed not only during mouse and human embryogenesis but also from mouse postnatal development to maturity, and this study would offer more new clues for further studing this gene function. 2. The level of NDRG2expression was detected in various tumor specimens, and further analyzing whether the cause of NDRG2 low-expression in those tumor specimens was relative with the gene mutation as that of inactivation of most tumor suppressor genes. 3. Using the tumor cell lines and normal neuron model cells as target to study the affection of NDRG2 expression on the growth of cells, and further investigate the possible mechanisms.[METHODS] 1. RT-PCR was used to detect the expression level of NDRG2 mRNA in whole mouse embryos from E8.5 to E17.5. Using the anti-NDRG2 monoclonal antibody (McAb), the serial sections of mouse whole embryos between E9.5 and E17.5 were analyzed by immunohistochemistry and immunofluorescence. Immunohistochemistry was used to analyze the expression partern of NDRG2 and N-myc. By immunohistochemistry, the expression level of NDRG2 protein in a large set of tissues from human embryos were analyzed. 2. Immunohistochemistry was used to analyze the expression trend of NDRG2 protein in heart, liver, brain, kidney and skeletal muscle during mouse postnatal development from PO to P28. Western blot and immunohistochemistry were used to analyze the expression distribution of NDRG2 protein in the various tissues from adult mouse. 3. RT-PCR and Western blot were used to detect the expression level of NDRG2 mRNA and protein in human fresh tumor specimens (including glioma, liver cancer and pancreatic cancer) and corresponding para-cancerous normal tissues collected from clinical operation, and then data was analyzed by statistics software. Using PCR-SSCP silver staining to detect whether there are the point mutation of those tumor specimens of NDRG2 low-expression confirmed by RT-PCR and Western Blot. 4. RT-PCR and Western Blot were used to detect the expression level of NDRG2 in eight kinds of cell lines. Using recombinant plasmid pcDNA3.0-NDRG2 to transfect MCF7 cells of NDRG2 low-expression confirmed RT-PCR and Western Blot, and the recombinant plasmid pSilencer3.1-NDRG2 was used to transfect PC 12 cells of NDRG2 high-expression confirmed RT-PCR, further the transfection efficiency weredetected by RT-PCR and Western Blot. After transfection the morphological change of cells were observed by phase contrast microscope, MTT was used to assay the change of cellular growth, Flow Cytometry was used to determine cell cycle, and DNA Ladder was used to analyze cellular apoptosis. [RESULTS] 1. The total trend of NDRG2 mRNA expression became higher in whole mouse embryos from E8.5 to El7.5. Before El2.5, NDRG2 immunoreaction was quite low in whole mouse embryo, and prominent immunoreactions were mainly detected in the developing heart. After 12.5, NDRG2 immunoreactions could be seen in the development of tissues and organs, including the heart, brain, lung, gut, liver, kidney, skeletal muscle, cartilage, chorion, and epidermis and whisker follicles. 2. At E9.5, only heart in whole mouse embryo could be seen the immunoreactions of NDRG2 and N-myc, and with increasing ages, NDRG2 immunoreactions in heart became stronger, but N-myc immunoreactions in heart became lower. After El4.5, the immunoreactions of NDRG2 and N-myc could be seen in superficial layer of epidermis, chondrocyte, epithelium of gut, skeletal muscle, epithelium of small bronchus, deciduas basalis of placenta and trophocyte of chorion. At same ages, NDRG2 immunoreactions in superficial layer of epidermis, trophocyte of chorion, skeletal muscle and chondrocyte, was stronger than N-myc immunoreactions, whereas N-myc immunoreactions in epithelium of gut, epithelium of small bronchus and deciduas basalis of placenta, was stronger than NDRG2 immunoreactions. 3. In human embryo tissues from four month and six month of gestation, NDRG2 immunoreactions could be seen in epithelium of small intestine, epithelium of large intestine, superficial layer of epidermis, whisker follicles, epithelium of small bronchus, hepatocyte, cardiac muscle cell, thymus corpuscles and epithelium of renal tubule, and NDRG2 immunoreactions in embryo tissues from six month of gestation, was stronger than that of four month of gestation. 4. During mouse postnatal development, NDRG2 immunoreactions in the heart, brain, liver, kidney and skeletal muscle, did nothave evident change between PO and P7, by contrast to the stages, NDRG2 immunoreactions in the heart, liver and skeletal muscle become slightly low, but in brain and tubule of renal cortex higher between P7 and P14. After PI4, NDRG2 immunoreactions in the brain (especially midbrain and cerebellum), heart and liver, became higher. 5. The result of western blot showed that anti-NDRG2 McAb could identify a single band at about 41 kDa on NC film, and the specific 41 kDa band was strong in the heart, liver and brain, moderate in kidney and weak in lung, pancreas and spleen. In various tissues from adult mouse, NDRG2 immunoreactions could be seen in cardiac muscle cell, brain, hepatocyte, epithelium of renal tubule, skeletal muscle, retina, crystalline lens, tongue(musculi linguae, filiform papillae and fungiform papillae), duct of sublingual gland, bronchial cartilage, adrenal cortex, interstitial cell of testis and epididymis, interstitial cells of ovary, epithelium of uterine glands. 6. Compared with corresponding para-cancerous normal tissues, the expression levels of NDRG2 mRNA in human glioma , liver cancer and pancreatic cancer reduced significantly (/><0.05); Using PCR-SSCP, mutation of the whole encoding region of NDRG2 was not found in these cancer tissues that the expression of NDRG2 reduced markedly. 7. Expression level of NDRG2 was low in MCF7 cells in eight kinds of cell lines detected by RT-PCR and Western Blot, but high in PC 12 cells. Compared with un-transfection or void plasmid group, the expression level of NDRG2 in MCF7 transected by recombinant plasmid pcDNA3.0-NDRG2 cells became high after transfection, whereas, the expression level of NDRG2 was reduced in PC 12 transected by recombinant plasmid pSilencer3.1-NDRG2. Through phase contrast microscope observation and MTT assay, it was found that the growth of MCF7 and PC 12 cells was inhibited. Cell analysis by FCM showed that proportion of MCF7 cells in Gl phase increased, and a typical sub-2N DNA peak (apoptosis peak) was observed in PC 12 cells, the percentage of apoptosis cells was obviously increased, compared with void plasmid group (?<0.05). After agarose gel electrophoresis, DNA ladder was...