K. Burgess,1 E. Benson,1 Z. Desta,1 A. Gaedigk,2 Y. Liu,1 T. Skaar1; 1Indiana University School of Medicine, Indianapolis, IN, 2Children's Mercy Hospital and Clinics, Kansas City, MO
BACKGROUND: miRNAs are predicted to regulate the expression of several CYP genes. However, these predictions need to be validated in cells that express the endogenous CYP genes. In order to induce the expression of these genes in cell lines, we have investigated the utility of TALE-TFs. TALE-TF proteins can be designed to induce the expression of any gene through a repeat variable diresidue code targeting sequences near the promoter region of the gene of interest and activates transcription by the attached VP64 transcription factor. Using this technique, we induced endogenous CYP2B6 expression and used it to study miRNA that target CYP2B6.
METHODS: We designed four TALE-TFs (GeneCopoeia™) to target CYP2B6 and transfected each individually and in combination into HeLa and HepG2 cells to determine how to maximize CYP2B6 induction. Subsequently, TALE-TFs were cotransfected into HeLa cells with miRNAs predicted to target CYP2B6 and compared to a C. elegans miRNA negative control. CYP2B6 mRNA levels were measured using quantitative PCR; expression levels were normalized to GAPDH.
RESULTS: Transfection of the combination of all four TALE-TFs in HeLa and HepG2 cells increased CYP2B6 expression 121- and 70-fold while transfections with individual TALE-TFs increased expression up to 18- and 50-fold compared to the CYP2B6 empty vector control. Hsa-miR-29a-3p was experimentally validated to target CYP2B6, reducing expression 2.4-fold (p=0.042).
CONCLUSION: We successfully designed TALE-TFs to induce CYP2B6 expression in HeLa and HepG2 cells. Reduction of CYP2B6 mRNA with hsa-miR-29a-3p suggests that this miRNA caused degradation of mRNA. These TALE-TFs should be a valuable tool for studying the molecular regulation of drug disposition genes.