Figure 3

TNF-α mRNA was a direct target of miR-181a-5p. (a) The mature sequences of miR-181 family members that arise from the 5′ arm of the precursors contain similar seed sequences (as shown in bold with underlining). Red color indicates the different nucleotides of each miR-181 family member. (b) The high scores of the conserved TNF-α 3′UTR as a computationally predicted target gene of miR-181a-5p. The mirSVR Score and the PhastCons score reflect the effect of the miRNA acting on mRNA and the conservatism of the predicted binding sites in the target mRNA, respectively. The 80 bp sequence of the TNF-α-3′UTR containing the predicted miR-181a-5p binding sites (the WT sense) and its mutant (the MUT sense) were synthesized for cloning into a pmirGLO Vector. (c) Dual-luciferase assay (based on the illustration of the pmirGLO vector multiple cloning site provided in the manufacturer’s instructions). Schematic of pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) used to construct TNF-α-3′UTR- based miRNA reporter plasmids. The dual-luciferase vector contains both modified firefly (hluc⁺) and Renilla (hRluc) luciferase genes. The desired miRNA recognition elements (MRE) were cloned into SacI-XhoI sites in the multiple cloning site (MCS) located in the 3′UTR of the hluc gene. (d) Luciferase activity in 293 T cells transfected with four types of vectors including TNF-α-pmirGLO (WT) or mutant of TNF-α-pmirGLO (MUT), two duplicates of the miR-181a-5p inhibitor-pmirGLO (PC) and empty pmirGLO (NC) plus mimic NC and mmu-miR-181a-5p. Results of 3 independent experiments were shown as the mean ± SEM. ***P < 0.001 vs. mimic NC group.