ABSTRACT
Cell Culture, and Xenografts ......................................................... 45 3.2.2 DNA Purification and Sodium Bisulfite Conversion ........... 46 3.2.3 Pyrosequencing Assay ......................................................... 46 3.2.4 Methylation-Specific PCR ................................................... 47 3.2.5 RNA Extraction, cDNA Synthesis, and qRT-PCR .............. 47 3.2.6 Flow Cytometry ................................................................... 47 3.2.7 Chromatin Immunoprecipitation .......................................... 48 3.3 Discussion ...................................................................................... 48 3.4 Results ............................................................................................ 51 3.4.1 The CD133 Promoter Region is Hypermethylated in
Prostate Cancer Cell Lines ............................................................. 51 3.4.2 CD133 Expression is Regulated by DNA Methylation in
Prostate Cell Lines ......................................................................... 52 3.4.3 Low Levels of CD133 Promoter Methylation are Found
in Prostate Tissues and Primary Epithelial Cultures ...................... 56 3.4.4 CD133 Expression is Not Regulated by DNA
Methylation in the Prostate Epithelial Hierarchy .......................... 61 3.4.5 Chromatin Structure, Together with DNA
Hypermethylation, Lead to CD133 Downregulation in Prostate Epithelial Cells ............................................................................... 63
3.5 Conclusion ..................................................................................... 66 Keywords ................................................................................................ 67 Acknowledgment .................................................................................... 67 Competing Interests ................................................................................ 67 Authors’ Contributions ............................................................................ 67 References ............................................................................................... 68 Credits ..................................................................................................... 70
3.1 INTRODUCTION
Epigenetic regulation of gene expression is a dynamic mechanism, which permits precise regulation throughout differentiation [1]. It plays a crucial role in preserving the hierarchical structure of tissues and is involved in maintaining stemness and fate determination of adult stem cells [2, 3]. Indeed, DNA methylation varies throughout cell differentiation [4] and epigenetic control is required for the multipotency of hematopoietic stem cells [5].
