ER~ is one of two estrogen receptors (ERs) which mediates estrogen action. ER~ is often down regulated in cancer compared with normal cells, but the molecular mechanisms involved remain unclear. The aims of this thesis were to examine whether ER~ and its splice variants are transcriptionally silenced by DNA methylation and post- transcriptionally regulated by miRs. The methylation status of the ER~ promoters (OK, ON and E1) and untranslated exons (OK and ON) were analyzed using bisulphite sequencing PCR (BSP) and methylation-specific PCR (MSP), in a panel of breast cancer cell lines of different ER~ status and clinical samples. The expression of ER~1, -~2 and -~5 mRNA was determined using (RQ) RT-PCR and correlated with the methylation data. ER~1 and -~2 expressions were inversely correlated with the methylation status of exon ON. However, no correlation was observed between promoters OK, ON and E1, and exon OK, ER~1 and -~2 expressions. ER~5 was abundantly expressed in these samples and did not correlate with the methylation of promoters and untranslated exons, suggesting that ER~5 was not regulated by DNA methylation. Pharmacological restoration of ER~ 1 and - ~2, but not -~5 mRNA, after in vitro DNA demethylation and histone deacetylation, using 5-aza-dc and TSA, provided experimental evidence that ER~1 and -~2 expressions were epigenetically regulated. microRNAs (miRs) are a class of non-protein coding small RNAs that regulate the expression of genes at post-transcriptional levels. In silico analysis using the miRGen Targets, MicroCosm Targets and RNA hybrid predicted a putative miR-92 target sequence within the 3'-untranslated region (UTR) of II ER~1. Expression analysis in breast cell lines and in a cohort of primary breast tumours confirmed a significant negative correlation between miR-92 and both ER~1 mRNA and protein, but not -~2, -~5 and ERa. Inhibition of miR-92 in MCF-7 cells increased the ER~1 expression in a dose-dependent manner, whereas miR-92 over expression led to ER~1 down-regulation. Reporter constructs containing candidate miR-92 binding sites in the 3'UTR of ER~1 confirmed that miR-92 down-regulated ER~1 via direct targeting of its 3'UTR. miR-92 expression was also modulated by the ER ligands 17~-estradiol and tamoxifen in MCF-7 cells. In summary, this study has provided new mechanistic insight on possible mechanisms that are. responsible for the down-regulation of ER~ in breast cancer. Besides the involvement of DNA methylation in regulation of the ER~ expression, this study has provided, for the first time, novel evidence that demonstrates the involvement of miR-92 in ER~1 regulation. These findings could provide the basis for potential therapeutic strategies for breast cancer, aimed at reactivating the expression of ER~1 through the manipulation of miR- 92 expression.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:555850 |
| Date | January 2010 |
| Creators | Al-Nakhle, Hakeemah Hussein Badi |
| Publisher | University of Leeds |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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