In our recent study, we found that miR-141-3p and miR-429b-3p have higher expression level in XY than YY testis [23 (link)]. To investigate potential targets of miR-141-3p and miR-429b-3p, first the Open Reading Frame (ORF) and 3’UTR of YY highly expressed unigenes were predicted, by searching against the vertebrate genomic database in GENSCAN (http://genes.mit.edu/GENSCAN.html ). Perl scripts of both Target Scan and miRanda were performed for searching the putative targets with default parameters, including context score percentile ≥100 for Target Scanand Max_Energy≤ −20 and for miRandabased on hybrid energy and stability [44 (link),45 (link)]
To characterize the interaction between miR-141-3p/-429b-3p and their predicted target genes, the 3’-UTR of selected putative target genes (Itgb2 and Gria4a) were inserted into the pmirGLO expression vector (Promega, USA). Hek-293T cells were seeded in 96-well plates and co-transfected with the constructed vectors and microRNA mimics or its control oligonucleotide using DharmaFECT transfection reagent (Dharmacon). 36h post transfection, the dual-luciferase reporter assay system was used to detect reporter (Firefly and Renilla) activity as described [46 ]. The profile of relative luciferase activities were normalized to Renilla luciferase activities.
To characterize the interaction between miR-141-3p/-429b-3p and their predicted target genes, the 3’-UTR of selected putative target genes (Itgb2 and Gria4a) were inserted into the pmirGLO expression vector (Promega, USA). Hek-293T cells were seeded in 96-well plates and co-transfected with the constructed vectors and microRNA mimics or its control oligonucleotide using DharmaFECT transfection reagent (Dharmacon). 36h post transfection, the dual-luciferase reporter assay system was used to detect reporter (Firefly and Renilla) activity as described [46 ]. The profile of relative luciferase activities were normalized to Renilla luciferase activities.
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