The exact biological function of NDRG2 still needs to be clarified, but evidence is clearly pointing to, that the function relates to some kind of maintenance of normal cellular identity. Therefore, clarifying the connection between expression levels in normal and abnormal cells, in addition to its regulation, will be of great importance in elucidating the implication and function of this gene. In this thesis, using the human cancer cell lines, HCT116 and SW480, as a model system, it was shown that NDRG2 is significantly down-regulated in both cancer cell lines, when compared to expression levels in normal human liver and that this expression profile correlated with an undetectable level of NDRG2 protein in the HCT116 cancer cell line. Further, it was observed that the NDRG2 promoter region was excessively methylated in both cancer cell lines, when compared to the methylation status of normal human genomic DNA. These observations taken together, indicates that the expression profile observed for the cancer cell lines is caused by gene repression, due to excessive DNA methylation at the promoter region. This correlation was additionally analyzed and it was confirmed that the reduced levels of NDRG2 in the HCT116 cell line could be restored by treatment with the demethylating agent, 5-azacytidine. Although this restored expression clearly indicates that 5-azacytidine can reverse the methylation status of the NDRG2 promoter, this will have to be confirmed by for example MSP or sequencing of the 5-azacytidine treated cells. In addition, it could also be relevant to investigate if the restored NDRG2 level is also reflected at protein level, which for example could be analyzed by western blotting of the 5-azacytidine treated cells.
The NDRG2 promoter was also evaluated for additional regulatory mediators and it was shown that the MYC transcription factor was capable of binding the NDRG2 promoter in HCT116 cancer cells, which could indicate its involvement in the transcriptional repression of NDRG2 in cancer cells. Although the NDRG2 promoter was shown to be methylated, which provides the further possibility of repression by methyl-binding proteins, the MeCP2 was not shown to bind the methylated NDRG2 promoter in HCT116 cancer cells. However, it was in fact not analyzed if that the cells did actually expressed the MeCP2 protein or confirmed that the antibody used was capable of binding MeCP2 and therefore further experiments will be required, in order to establish if the MeCP2 is capable of binding to the NDRG2.
Although the experiment conducted in this master thesis, have provided some results on how NDRG2 may be epigenetically regulated in cancer, there is still a lot to be learned on the topic. In addition to
the further studies on the transcriptional repression caused by DNA methylation, it could be relevant to analyze the binding of the methyl-binding proteins. Since the methyl-binding proteins constitutes a large family, of which any in theory could bind and repress NDRG2, it should be investigated if any of the other methyl-binding proteins are in fact binding and participating in the observed down- regulation of NDRG2. In addition, could any the other epigenetic modifications also be involved in the down-regulation of NDRG2 and it could therefore be interesting to investigate if the NDRG2 is also regulated by for example aberrant histone modifications, abnormal ATP-dependent chromatin remodeling or ncRNAs.
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