title: Human L-DOPA decarboxylase mRNA is a target of miR-145: A prediction to validation workflow.
abstract: l-DOPA decarboxylase (DDC) is a multiply-regulated gene which encodes the enzyme that catalyzes the biosynthesis of dopamine in humans. MicroRNAs comprise a novel class of endogenously transcribed small RNAs that can post-transcriptionally regulate the expression of various genes. Given that the mechanism of microRNA target recognition remains elusive, several genes, including DDC, have not yet been identified as microRNA targets. Nevertheless, a number of specifically designed bioinformatic algorithms provide candidate miRNAs for almost every gene, but still their results exhibit moderate accuracy and should be experimentally validated. Motivated by the above, we herein sought to discover a microRNA that regulates DDC expression. By using the current algorithms according to bibliographic recommendations we found that miR-145 could be predicted with high specificity as a candidate regulatory microRNA for DDC expression. Thus, a validation experiment followed by firstly transfecting an appropriate cell culture system with a synthetic miR-145 sequence and sequentially assessing the mRNA and protein levels of DDC via real-time PCR and Western blotting, respectively. Our analysis revealed that miR-145 had no significant impact on protein levels of DDC but managed to dramatically downregulate its mRNA expression. Overall, the experimental and bioinformatic analysis conducted herein indicate that miR-145 has the ability to regulate DDC mRNA expression and potentially this occurs by recognizing its mRNA as a target.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Athens, Athens 15701, Greece.
authors.forename: Emmanuel I
authors.initials: EI
authors.lastname: Papadopoulos
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Athens, Athens 15701, Greece.
authors.forename: Emmanuel G
authors.initials: EG
authors.lastname: Fragoulis
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Athens, Athens 15701, Greece. Electronic address: ascorilas@biol.uoa.gr.
authors.forename: Andreas
authors.initials: A
authors.lastname: Scorilas
mesh_terms: D000465:Algorithms; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D019295:Computational Biology / Q000379:methods*; D004296:Dopa Decarboxylase / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D054928:Validation Studies as Topic
chemical_list: C541010:MIRN145 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D004296:Dopa Decarboxylase; D001142:Aromatic-L-Amino-Acid Decarboxylases; C000595218:DDC protein, human
keywords: Experimental validation; MicroRNAs; Real-time PCR; Target prediction algorithms; l-DOPA decarboxylase