Recently, the importance of non-coding regions of the genome, which were once presumed to be “junk” DNA, has been revealed. The non-coding 3’ untranslated region (3’UTR) plays a significant role in the regulation of microRNA (miRNA) functions. The 3’UTR is hypothesized to function in the feed-back regulation of miRNA functions, since it can bind and inactivate multiple miRNAs. In this study, the effects of the exogenous over-expression of two 3’UTRs, CD44 and Tumour Suppressor candidate 2 (TUSC2), on miRNA functions in breast cancer carcinogenesis were investigated. A series of cell function assays in human and mouse breast cancer cell lines, MT-1, MDA-MB-231 and 4T1 showed phenotypic changes caused by the 3’UTRs. A variety of cell function characteristics were affected, including cell proliferation, colony formation, cell survival, angiogenesis, tumour growth, cell migration, invasion and adhesion. These results were hypothesized to occur due to the interaction of the 3’UTRs with multiple miRNAs. The 3’UTRs were able to antagonize cytoplasmic miRNAs, as demonstrated by luciferase activities. In the case of the CD44 3’UTR, downstream target mRNAs, CDC42, Col1a1 and FN1 had an increased expression along with CD44. When the 3’UTR of TUSC2 was over-expressed, there was an increased translation of TUSC2 and the downstream targets, tissue inhibitor of metalloproteinases (TIMP) -2 and -3.The 3’UTRs of these target mRNAs can bind and target multiple miRNAs in common with the TUSC2 3’UTR, which were confirmed with luciferase activity assays and correlated with a series of siRNA and miRNA assays. During the study of the TUSC2 3’UTR, a potential pseudogene, TUSC2P, was discovered. Interestingly, TUSC2P mRNA expression was found to be decreased in cancer cells compared to normal cells and had similar cell functional characteristics as that of the TUSC2 3’UTR. These findings suggest that the over-expressed non-coding transcript can serve as a competitor for miRNA binding, which freed the potential targets of the miRNAs and led to an up-regulation of multiple protein levels. The non-coding transcript can thus be used as a functional miRNA inhibitor that is capable of modulating multiple miRNAs, which can be applied towards treating cancer in the form of gene therapy.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/65479 |
| Date | 19 June 2014 |
| Creators | Jeyapalan, Zina |
| Contributors | Yang, Burton |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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