Dual Luciferase Reporter Gene Assay

According to starbase (http://starbase.sysu.edu.cn/index.php) (Li et al., 2014), there are binding sites between miR-186 and TLR4. The specific binding fragment and its mutant (MUT) were cloned into pmiR-GLO dual luciferase vector (firefly and renilla) (Promega, Madison, WI, United States), and recorded as wild-type (WT)-TLR4 and MUT-TLR4. In accordance with the instructions, Lipofectamine^TM 2000 (Invitrogen Inc., Carlsbad, CA, United States) was utilized to co-transfect NC mimic and miR-186 mimic into 293T cells (China Center for Type Culture Collection, Wuhan, Hubei, China). Luciferase activity (firefly and renilla) was detected 48 h later using Dual-Luciferase Reporter Assay System (Promega).