Cancer is a leading cause of death worldwide. This disease is nowadays considered to be caused by both genetic and epigenetic alterations, which through activating oncogenes and/or inactivating tumor-suppression genes, leads to an uncontrolled cell growth and division. Although the involvement and function of genetic changes in cancer are well established, the field of cancer epigenetics is still relatively new and there is yet a lot to be learned about the involvement and function of epigenetic changes in cancer. One of the more thoroughly studied epigenetic changes in cancer is that of excessive DNA methylation at promoter regions, which has been linked to the silencing of for example tumor-suppressor genes. Therefore, in order to contribute to the understanding and elucidation of how genes may be epigenetically regulated in cancer, I investigated the recently accepted tumor-suppressor, NDRG2, which has been shown to be down-regulated for many cancers and to be subjected to excessive DNA methylation at the promoter region, using the cancer cell lines HCT116 and SW480. The NDRG2 expression was analyzed by Quantitative Real Time Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) and experiments showed that NDRG2 levels were significantly down-regulated in both cancer cell lines. Further, the NDRG2 protein level was analyzed by western blotting for the HCT116 cells and results showed that NDRG2 was also down-regulated at protein level in this cell line. Additionally, the NDRG2 promoter was examined by Methylation specific Polymerase Chain Reaction (MSP) and sequencing, which showed that the previously observed NDRG2 down-regulation also correlated with an excessive DNA methylation at the promoter region in both cancer cell lines. Therefore in order to further investigate the correlation, the HCT116 cells were treated with the demethylating agent, 5-azacytidine, and analyzed by qRT-PCR. This revealed that the cells treated with 5-azacytidine had a significantly increased NDRG2 expression, when compared to untreated cells. The promoter of NDRG2 was also investigated by Chromatin Immunoprecipitation (ChIP) for the binding of MYC and MeCP2, and the result was analyzed by Quantitative Real Time Polymerase Chain Reaction (qPCR), to see if these proteins contributed to the down-regulation of NDRG2 expression. The results showed that MYC was capable of binding to the NDRG2 promoter, but that the MeCP2 was not. In conclusion, these results strongly indicate that the down-regulation of NDRG2 observed for both cancer cell lines and the down-regulation of NDRG2 protein shown for the HCT116 cells, is caused by the methylation of the NDRG2 promoter region and this notion is in fact supported by the 5-azacytidine being capable of restoring the NDRG2 expression in the HCT116 cells. In addition, it seems like MYC is also involved in regulating NDRG2, but this relation will have to be further investigated.
|Uddannelser||Molekylærbiologi, (Bachelor/kandidatuddannelse) KandidatMedicinalbiologi, (Bachelor/kandidatuddannelse) Kandidat|
|Udgivelsesdato||27 jan. 2014|
- N-myc Downstream Regulated Gene 2
- expression level
- Western blotting
- Cell line SW480
- Cell line HCT116