The Relationship Between NDRG2and P53 During DNA Damage And The Funcion Of NDRG2 In The P53 Signal Pathway

DNA is the most important genetic material in the living creature and the integrity and stablity of genomic DNA is very meaningful for the live and the normal physical activity of cells. But the genomic DNA is faced with the attack from vivo or vitro and can be damaged by the various factor in or out the cells. The genomic integrity of the organism is dependent on two systems which are independnt but intimate: DNA repair and apoptosis. DNA damage can promote one signal which can be accepted by the celluar cell cycle or apoptosis system. P53 is the most important transcriptional factor in the signal pathway of the DNA damage mediate, some target gens of P53,such as P21 ,14-3-3,GADD45 and Bax ,are invovled in the cell cycle arrest and apoptosis after the DNA damage and is important for the suppression of cancer.NDRG2 (also named SYLD/KIAA1248),a novel gene that down-regulated in glioma,was first discovered by our laboratory in 1999 with polymerase chain reaction ( PCR )-based subtractive hybridization when looking for the differentially expressed genes between glioma and normal tissue (the GenBank accession No .AF159092), the entire mRNA of NDRG2 is 2024 bp in length and it encodes a deduced 357 amino acids unkown protein with a calculated molecular mass of 41 kDa. The NDRG2 gene is located at chromosome 14q11.2, including 16 exons and 15 introns. For the highly homologous in amino acids to N-myc downstream-regulated gene 1(NDRG1), it was named NDRG2.Although a lot of works had been done on NDRG2 gene in recent years, its function is still not very clear. Our previous study showed that one kind of DNA damage reagent -Adr could induce the expression of NDRG2 in A549 cells, suggested that NDRG2 is involved in the DNA damage. P53 is the most important transcriptinoal factor, in additon, one putavie P53 binding site was found in the NDRG2 intron 1 through the bioinformatic method, and the other NDRG family gene-NDRG1 is necessary for P53 mediated apoptosis as a target gene of P53, the above information suggested that NDRG2 may be a target gene of P53 and may be involved in the P53 signal pathway.To verify the above hypothesis, we firstly found that the NDRG2 mRNA and protein level were increased after the P53 wild-type cell line (A549 and U87) were treated with Adr for different period by real-time PCR and Western blot, however the expression level of NDRG2 have no change in the P53 mutant (MDAMB-231and SK-BR-3) or P53 deleted cell lines (H1299) with Adr treatment. So the above results suggested that the increasing of NDRG2 expression level was dependent on P53 during DNA damage.Then, to further ensure the relationship between P53 and NDRG2, the P53-deleted cell line H1299 was over-expressed exougenous P53 and the expression level of NDRG2 was increased with the increased P53 expression level. We also found that P53 can specifically not only up-regulate but also bind to the P53 binding site in the NDRG2 intron1 by the luciferase assay, ChIP and EMSA. So the above results suggested that NDRG2 is a target gene of P53 and provide the clue for the study of NDRG2 function.Finally, we further study the role of NDRG2 on the P53 signal pathway. We found that the RNAi of NDRG2 obviously suppressed the apoptosis induced by P53 overexpression and suggested that NDRG2 is important for the P53 mediated apoptosis. In additon, the over expression of NDRG2 suppressed the proliferation and increased the chemosensitivity of the cancer cells with chemical regent -Adr and cisplatin. So the above results indicated that NDRG2, as a new P53 target gene, is necessary for the P53 mediated apoptosis and can obviously increase the chemosensitivity of some caner cells.From all the above results, we conclude that NDRG2 is a new target gene of P53 and involved in the response to DNA damage, it is necessary for the P53 mediated apoptosis and can increase the chemosensitivty of some cancer cells. So our study not only improved the P53 signal pathway but also provide some meaninful clue and verification for the therapy of caner.The final part is some of my master research content. The aim of this part is to analyze the mechanism invovled in the NDRG2 down-regulated in HepG2 cells. Using single strand conformation polymorphism (SSCP) and direct sequencing analysis of the PCR products, we identified one mutant site,–13 bp (C>T), in the NDRG2 promoter region from the HepG2 genome. In addition, the transcriptional activity of the alternative promoter was significantly decreased when compared to the normal promoter as measured using the luciferase activity assay.