Since the initial description of cancer stem cells (CSCs) as a self-renewing subpopulation of malignant cells with tumor-initiating capacity, a growing body of evidence has supported the existence of CSCs in virtually every tumor type. Our previous work in colorectal cancer has identified the transcription factor CDX1 as a key regulator of colorectal CSC differentiation. CDX1 expression is frequently lost in colorectal cancer, resulting in more aggressive, poorly differentiated tumors with higher proportions of CSCs. Many miRNAs have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in colorectal cancer, remain poorly understood. We began by identifying miRNAs downstream of CDX1 by using high-throughput small-RNA sequencing to profile miRNA expression in two pairs of colorectal cancer cell lines with stable CDX1 overexpression or knockdown. Validation of candidates identified by RNAseq in a larger cell line panel revealed miR-215 to be most significantly correlated with CDX1 expression. ChIP-qPCR and promoter reporter assays confirmed that CDX1 directly transactivates miR-215 transcription. MiR-215 is depleted in FACS-enriched CSCs compared to unsorted samples. Overexpression of miR-215 in poorly-differentiated, highly clonogenic cell lines causes growth arrest and a dramatic decrease in colony formation. miR-215 knockdown using a miRNA sponge causes an increase in clonogenicity and impairs differentiation in CDX1-high cell lines. Indeed, the effects of CDX1 expression on both gene expression and colony morphology can be attenuated by miR-215 inhibition, indicating that miR-215 is a functional mediator of CDX1. Microarray studies following miR-215 overexpression indicate that miR-215 induces terminal differentiation-associated growth arrest, due in part to direct silencing of BMI1 expression and de-repression of BMI1 target genes including CDKN1A. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in colorectal cancer. We further characterize another miRNA-transcription factor axis in colorectal cancer, and we identify the novel miR-3189-3p as a potent effector of cell death with potential therapeutic implications.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:627861 |
| Date | January 2014 |
| Creators | Jones, Matthew |
| Contributors | Bodmer, Walter F. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:5462857b-4cee-44a8-a299-63b798a67f3f |
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