P-71: Construction of Required DNA Plasmids for Validation of Predicted MicroR-NA Targets

Background: The micro-ribonucleic acids (miRNAs) are noncoding RNA molecules that are conserved developmentally and include usually 18-25 nucleotides. MiRNA regulates gene expression through mRNA degradation or inhibiting of its translation. These biomolecules contribute in cellular physiologic and pathologic processes and most of them may act as oncogenes or tumor inhibitors. Identification of miRNAs target sequences is critical for investigation of their physiology; and in this study it is considered to establish two constructs as model, for validation of bioinformatically predicted targets for miRNAs in the future. Materials and Methods: In the present study, two expression vectors were constructed.The first one (vector A) expresses precursor of mir-101 under control of CMV promoter. The other vector contains coding strand of EGFP and Luciferase fused to each other as a reporter gene downstream of elongation factor promoter. In this vector 3’UTR region of the gene of ATP5B (as target of mir-101) was cloned between transcription terminator sequence and the poly A region. For assessment of the functionality of these two vectors, a stable cell line was obtained by transfection of HeLa cells by vector B and next, mir-101 was over-expressed in the aforementioned cell line using vector A. Difference in expression of the reporter gene was assessed after transfection of the cell line by vector A. Results: Down regulation of the reporter gene confirmed the suitability of this system. Conclusion: It was concluded that this system is reliable for validationof other predicted targets of any miRNA by substitution of the precursor of the mir of interest in the vector A and cloning of the related 3’UTR region in vector B.