Creation of a Dual-Fluorescence Reporter System to Assay miRNA Regulation of APOBEC3A

MicroRNAs have an important role in regulating gene expression, including regulating the transcripts of the APOBEC3 family of cytidine deaminases. The APOBEC3 family is part of the immune defense against viral infection but has also been implicated in cancer progression. The hypothesis for this thesis is that specific miRNAs could be identified that regulate APOBEC3A (A3A) expression. I used molecular cloning and confocal microscopy to evaluate the miRNA-mediated silencing of A3A. A commercially available vector, psiCHECK-2, can be used to assay microRNA (miRNA) activity on specific targets. Molecular cloning was performed to modify the psiCHECK-2 Dual luciferase system (Renilla and Firefly) into a Dual fluorescence system (mCherry and eGFP). In the Dual fluorescence construct, the mCherry transcript is fused to the experimental 3'-UTR, while eGFP is expressed independently, enabling analysis of mCherry fluorescence as a ratio of eGFP fluorescence to assay for miRNA mediated silencing. To validate this new construct, the 3'-UTR of eIF2S3, which encodes a translation initiation factor, was cloned into the multiple cloning site of the Dual fluorescence construct and co-transfected with a plasmid expressing hsa-miR-1285, which has been described as robustly silencing eIF2S3. Our confocal analysis showed a significant decrease in mCherry: eGFP fluorescence mediated by hsa-miR-1285. Finally, we cloned the 3'-UTR of A3A into the Dual fluorescence construct and individually co-transfected seven different miRNAs, including hsa-miR-1285, which were predicted to silence A3A. Our confocal analysis demonstrated a significant decrease in mCherry: eGFP fluorescence for each of the tested miRNAs, compared to a control group with no miRNA and a non-silencing control, hsa-miR-527, indicating that the predicted miRNAs mediate silencing of A3A by interacting with the 3'-UTR of the transcript. The interaction between miRNAs and A3A is not well studied, and we identified multiple miRNAs that could play a role in silencing A3A. These data could be used for future development of anticancer therapeutics that target A3A.