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. 2014 Feb 20;15(2):2971–2990. doi: 10.3390/ijms15022971

Figure 7.

Figure 7.

Luciferase reporter assay for binding of the modified ERH-3′-UTR with miR-574-3p. (A) The miR-574-3p target sequence was predicted to be at nucleotide positions 427 to 446 (3′-UTR) in ERH [Genbank: NM_004450.2]. The putative miR-574-3p recognition sequence (solid lines) was cloned with four repetitions downstream of the firefly luciferase gene in the pMIR-REPORT vector. Expression of the luciferase gene is controlled by the CMV promoter and SV40 polyA; (B) A549 cells were transfected with the pMIR-ERH plasmid or pMIR-REPORT vector without the ERH sequence as a control at 25 pM and 6 h later with synthetic miR-574-3p or the miR-574-3p antagomirs at 10 nM. Luciferase activity was measured 6 h after transfection with the synthetic miR-574-3p or miR-574-3p antagomirs. Each experiment was conducted three times. Error bars represent the standard deviation. * p < 0.05 vs. cells transfected with the pMIR-ERH vector.