UBC Theses and Dissertations
Identification of a novel microRNA involved in non-specific binding to a decoy transcript Slowski, Kathryn Johanna
MicroRNAs are known to be upregulated or downregulated in various types of cancer, leading to changes in the expression of genes involved in cellular proliferation, anti-apoptosis, migration, and invasion. To study the effects of microRNA loss or gain in different neoplasms, numerous models have been described to decrease or increase expression of microRNAs, but the off-target effects of different methods have not been well investigated. I investigated the possibility of off-target effects in a model of miR-143 knockdown in myeloid leukemia cell lines that implemented a microRNA sponge, or decoy, as a method to reduce microRNA expression. The high expression of a sponge with repetitive sequence elements and low expression of the intended microRNA for knockdown, miR-143, created conditions with increased potential for non-specific microRNAs to bind to the sponge. Therefore, I investigated the potential binding sites present in the sponge and whether any novel microRNAs could bind to these sites. I found a number of potential candidates and eliminated them based on their likelihood of regulating protein targets and their resemblance to a microRNA in structure, leaving one potential candidate. I found genomic evidence of the existence of this novel microRNA, evolutionary conservation of function, and performed assays that confirmed the biological activity. Next, the original sponge was redesigned to inhibit the binding of the potential non-specific microRNA; miR-X, or the miR-143 binding sites were mutated to inhibit the binding of miR-143 and capture miR-X instead. This demonstrated that binding of non-specific microRNA could be abrogated and differential protein abundance specific to the knockdown of each microRNA separately was verified. I conclude that non-specific binding to the sponge is a distinct possibility in experiments using this method of microRNA knockdown, which needs to be taken into account when designing sponges in the future. This work also demonstrates that there remain novel microRNAs awaiting discovery.
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