| Our group discovered the ndrg2 gene (N-myc downstream regulated gene 2) and cloned it from normal human whole brain cDNA library in 1999, which has been adopted in Genbank with the number of AF159092. This gene locates on human chromosome 14q11.2. Its full mRNA is 2,024bp in length, encoding about 40kDa protein with 357 amino acid. The ndrg2 gene is one member of ndrg gene family, which consists of 4 members: ndrg1, ndrg2, ndrg3 and ndrg4. This gene family has high similarity in nuclear acid or amino acid sequence from inferior creature to advanced creature. These indicate that the ndrg gene family plays an important role in life of cells. Our preliminary studies showed that there were higher expression of ndrg2 mRNA in brain, salivary gland, skeletal muscle, lower expression of ndrg2 in bone marrow, testis, peripherial blood and placenta and none expression in bone marrow cells (BMC) of leukaemia patients, colorectal cancer tissue, lung cancer tissue, liver cancer tissue and some tumor cell lines. In addition, the ndrg2 gene could inhibit the proliferation of many tumor cells, such as glioma cell line BT325. All above suggested that gene might be a new candidate tumor suppressor gene (TSG). However, the mechanism on suppressive effect of ndrg2 isn't clear.Therefore, to study its function and the relationship of signal transduction has important theoretical meaning. There have been no reports about the precise cellular and molecular function of ndrg2 gene. Our group aimed to establish a glioma cell line with ndrg2 gene steady expression and analyze the cellular characteristic, so as to investigate the ndrg2 gene better for future.First, semiquantitative RT-PCR analysis was utilized to observe the different expression of ndrg2 gene in A549, 293, sk-br-3, HT29, U251, U87 and SGC-7901. The result indicated that there were many difference in these cancer cell lines. The expression of the ndrg2 gene in A549, HT-29, U251 and 7901 were lower than which in 293 cell line. None expression was detected in sk-br-3 and U87 cell line. We selected U251 as the target cell line because of the lower expression and common use.After the pEGFP-c2-ndrg2 vector has been successfully constructed, it was transfected into U251 cell line. We use U251 cells without transfection as negative control, U251 cells transfected with pEGFP-c2 vector being transfected control. Following G418 screening, the surviving cell clone was elected and enriched. Then expression of the ndrg2 gene was detected by RT-PCR. We also observed expression of ndrg2 protein by western blot. Since the pEGFP-c2-ndrg2 vector contains green fluorescene, the expression of ndrg2 was also detected under fluorescent microscope. All above verified that the transfection was successful.For further investigation on the biological behaviors of U251 cells, we used flow cytometry, plate clone formation assay and MTT assay to investigate the characteristic of the transfected cells. The cell percentage of G1 phrase was about 73.2%, which is more than each control group. It indicated that the transfection of ndrg2 gene contributed to the inhibition of proliferation and induction of cell cycle arrest in G1 phrase. Compared with control group respectively, the curve of translated cells increased relatively slowly in MTT assay. The plate clone formation assay indicated that plate clone formation efficiency was lower than each control group (p<0.05). These proved that the steady overexpression of ndrg2 could inhibit the proliferation of glioma cells.We first established a glioma cell line with ndrg2 gene steady expression. Then we analyzed the cellular characteristic of transfected cells. The results proved that the proliferation, the growh rate and plate clone formation efficiency of transfected cells were slower than each control group. We concluded that the steady transfection of the ndrg2 gene could inhibit the proliferation of glioma cells, the same as the transient one. The result further proved that ndrg2 gene could inhibit proliferation of cancer cell lines, which can be selected as a candidate tumor suppressor gene (TSG). The investigation about the mechanism on suppressive effect of the ndrg2 gene and the exact signal transduction were settled for future. |
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