Overview of microRNA targeting

Introduction: MicroRNAs are post-transcriptional gene regulations. They function through binding in their target sequence in mRNAs. Thanks to significant effect of them, determining of their gene targets have shed light on molecular etiology of disease so would be useful in diagnosis and treatment. There are lots of bioinformatics databases like mirwalk (http://mirwalk.umm.uni-heidelberg.de/), mirTarBase (http://mirtarbase.mbc.nctu.edu.tw/), pictar (https://pictar.mdc-berlin.de/), TargetScan (http://www.targetscan.org/), which forecast target genes via matching of seed sequence of microRNAs in microRNA recognition element (MRE) and sequence of untranslated region of genes (UTRs). One of valid experiments to confirm this targeting is luciferase reporter gene assay test.Description: In-silico study to predict of possible targets of miRNA of interest were done. 3 UTR of mRNA consist of seed sequence were amplified from genomic DNA by PCR and was cloned into Psicheck construct, which expresses firefly luciferase by Xho1 and Not1 enzymes. HEK293 cells were seeded and co-transfected with 0.1 μg firefly luciferase reporter containing the gene 3'UTR and miRNA and mutant 3 UTR of gene as positive control, along with Renilla luciferase vector pRL-TK as an internal control using Lipofectamine 2000. All transfections were performed in two experiments, each in triplicate. Luciferase activities were measured according to the manufacturer’s instructions (Dual-Luciferase Assay System; Promega).Discussion and conclusion: Targeting of 3 UTR of gene of interest through miRNA led to an intense reduction of the luciferase signal. Mutations of miRNA binding sites in the 3′UTR reversed the inhibition and the signal of luciferase in positive and internal control is about equal. Use of bioinformatics and luciferase assay as tool for detecting of miRNAs targets can assist to determine the power of post-transcriptional regulators in targeted cell therapy by reduce or over expression of them to control their gene targets along with important molecular pathway in regenerative medicine field.