miRNA Regulation of A3A and A3B

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Gieseke, Katlyn; Lange, Alyssa
Enzymes and Coenzymes
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APOBEC3A (A3A) and APOBEC3B (A3B) are enzymes that deaminate DNA Cytosine to Uracil, leading to 5'TC to 5'TT mutations. A3B overexpression has been found in many different cancers and is associated with increased mutation. Some individuals have a deletion that removes the A3B coding region and fuses the A3B 3'regulatory region to A3A. If A3B is the sole source of these mutations, these individuals shouldn't have these APOBEC3 mutations, but they do. I hypothesize that the A3B deletion affects A3A expression through miRNA transcriptional regulation. To test this hypothesis, we amplified the A3A and A3B 3'regulatory regions from HCC1569 cancer cells, expressing high levels of A3B, and HeLa cells, expressing low levels of A3B. These regions of DNA from each cell will be cloned into psiCHECK-2 vector downstream of firefly luciferase. We then identified miRNA-'s that are predicted to bind the 3'regulatory region of A3A and A3B. We will clone the identified miRNA-'s into a separate expression vector. The luciferase vector with the A3A or A3B 3'regulatory region will be transfected into cells, along with a miRNA expression vector. The luciferase gene will cause the cells to glow. The light will be quantified to tell how the miRNA affects A3A and A3B expression. MiRNA that binds the regulatory region are expected to cause a decrease in luciferase. We expect to identify miRNAs that are capable of regulating A3A and A3B expression. This research could help identify how A3B expression is controlled and how cancer was caused in people with the A3B deletion. This research could also help to further understand how tumors and APOBEC enzymes are related.