Luciferase activity detection

For detecting NF-κB activity detection, HEK293T cells were co-transfected with pNF-κB-luc (1 μg) and pRL-TK (50 ng) plasmids in the presence of pPPE36 (1 μg) or control plasmid (1 μg) for 24 h using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Then, cells were treated with TNF-α (20 ng/ml) for 12 h. For detecting Jnk and p38 activation, HEK293T cells were co-transfected with pRacL61 (1 μg), pAP1-luc (1 μg), and pRL-TK (50 ng) in the presence of PPE36-Flag (1 μg) or control plasmid (1 μg) for 36 h. Cells were lysed and subjected to luciferase activity detection according to manufacturer instructions (Promega, Madison, WI, USA). All reactions were performed in triplicate.

The wild-type H19-3′UTR (WT), mutant H19-3′UTR (MUT), wild-type Sox6-3′UTR (WT) and mutant Sox6-3′UTR (MUT) containing the putative binding site of miR-19b were established and cloned in pmirGLO dual luciferase miRNA reporter vectors (Promega, Madison, WI, USA). The reporter vectors and miR-19b mimics or miR-NC were co-transfected into 293T cells using Lipofectamine 2000 (Invitrogen). After 36 h of incubation, cells were collected and lysed for luciferase activity detection (Promega).