Mechanism Studies Of Down-regulated Expression Level Of Candidate Tumor Suppressor Gene NDRG2 In Some Tumor Cell Lines And Breast Cancer Tissues

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. Genbank Accession number is AF159092.The NDRG family includes four homologues: NDRG1, NDRG2, NDRG3 and NDRG4, which are highly conserved protein in a variety of organisms, suggetsting their role in some important cellular process. The structured and functional research of NDRG2 is just beginning and our lab found that Ndrg2 was abundant in normal tissues. What' more, when NDRG2 gene was transferred into some tumor cells, the cellular proliferation would be partly suppressed and Ndrg2 protein in some tumor tissues was more than normal tissues, but the mechanism was not clear yet.In order to detect the mechanism about the down- regulated expression level of Ndrg2 in some tumor tissues. We firstly screened six kinds of tumor cell lines by RT-PCR. RNA dot blot, Western blot and immune- photochemical staining and obtained the cell lines that express none or less Ndrg2.Then we analyzed these cell lines separately by multiplex genomic PCR, PCR-SSCP, methylation sensitive restricted cleavage enzyme analysis to detect if there was LOH in NDRG2 genomic DNA, or methylation and point mutation in the promoter region.We screened the breast cancer cell lines , MCF-7, MDA-MB-231, SK-BR-3,stomach cancer cell lines, SGC-7901, BGC-823. MGC-803. lung cancer cell lines, A549, liver cancer cell lines HepG2 and glioblas-toma cell BT-325 as none or less Ndrg2 expression cell lines. Multiplex genomic PCR results showed that NDRG2 was LOH in MCF-7cell lines; but not in others. PCR-SSCP indicated that there was point mutation in the core promoter region of HePG2 cell lines. What" more, methylation sensitive restricted cleavage enzyme analysis suggested the possible promoter-methylation in SK-BR-3, BGC-823 and HepG2.Besides above, we also analyze the expression level of ndrg2 in 21 breast tumor tissues with matched normal tissues. 5 samples with down-regulated expression level of Ndrg2 were found, Fourthermore, we analyzed the mechanism of it by multiplex genomic PCR, PCR-SSCP.There was neither NDRG2 LOH nor core promoter point mutation in the 5 breast tumor samples.Based on our work, we get the following conclusions:Firstly, down-regulated expression level of Ndrg2 suggests the possible role of Ndrg2 in controlling the genesis or proliferation of cancer cells. The mechanism about the down-regulated expression level of Ndrg2 come from LOH in some cancer cells and point mutation or methylation of NDRG2 in others. In another word, the candidate tumor suppressor gene-NDRG2 inactive in several different ways both among different cancer cells and in one single cell line.Secondly, the down-regulated expression level of Ndrg2 in some breast tumor tissues ,may also indicate the genesis development or metastasis activity of Ndrg2 .Neither LOH nor point mutation of NDRG2 core promoter were detected in the 5 breast tumor tissue samples, which indicates that the NDRG2 LOH and the point mutation of NDRG2 core promoter may not be the main mechanism for down- regulated expression level of Ndrg2 in breast tumor tissue, it is speculated that there might be other mechanisms responsible for the down-regulated expression level of Ndrg2 .