Mechanisms Of Down-regulated Expression Level Of NDRG2 In Human Glioma

The human NDRG2 (N-myc downstream regulated gene 2) gene was first cloned from normal human brain cDNA library in our lab by anchored primer PCR. The Genbank Accession number was AF159092. Later, NDRG2 was cloned again with subtractive hybridization in glioma and adjacent normal tissues.The NDRG family includes four homologues: NDRG1, NDRG2, NDRG3 and NDRG4, which are highly conserved protein in a variety of organisms, suggesting their important role in some cellular process. The structure and function research of NDRG2 is just beginning. We have got that the expression of NDRG2 in lung cancer and hepatoma are lower than adjacent normal tissues. Moreover, when NDRG2 was transferred into some tumor cells, the cellular proliferation would be partly suppressed, but the mechanism is still not clear.In our study, we firstly analyzed the expression of NDRG2 mRNA in the gliomas and adjacent normal tissues by Real-time PCR. Secondly for further discussing the possible mechanism of NDRG2 down-regulation in glioma, BSP was used to examine the methylation status of NDRG2 promoter region in glioma tissues,adjacent normal tissues and glioma cell line.Besides, because the PI3K signal transduction pathway including AKT and PKC was remarkablely activated in glioma, and it was reported that NDRG2 was the substrate of AKT and PKC, so we detected the mRNA expression of these protein kinases, in order to get information about their interactions.Compared with adjacent normal tissues, the expression level of NDRG2 mRNA was reduced significantly in the glioma samples. And with the increase of pathologic grade, NDRG2 level had a trend of reduction. And the methylation rate of NDRG2 promoter region increased significantly in glioma tissue samples compared with adjacent normal tissue samples, and among three grades it was distinctly different. In addition,the methylation happened mainly in frontal 16CpG sites.There were more sites methylated in gradeⅣthan in gradeⅢ. The CpG islands of NDRG2 promotor region in U251 cell line was methylated, but not in SHG44 cell line.Besides, we analyzed the mRNA expression of PI3K, AKT2, PKC-αand found that only PKC-αwas increased significantly in glioma samples than in adjacent normal tissue samples, while the others were not obviously different in three grades. There was no statistical correlation between the level of mRNA expression of PI3K, AKT2 or PKC-αand that of NDRG2.It is necessary to study their relation in the level of protein.Based on our work, we get the following conclusions:The expression of NDRG2 gene was down-regulated in human glioma and the level of NDRG2 was negatively correlated with glioma grades. One possible mechanism of the down-regulation was aberrant methylation of NDRG2 promoter region and subsequent transcriptional inactivation.