Wild-type or mutant 3′-UTR of SUB1 were cloned into the pMIR-REPORT miRNA Expression Reporter Vector (Life Technologies). HEK293 cells were co-transfected with pre-miR-101 or controls and wild-type or mutant 3′-UTR-luc, as well as pRL-TK vector as an internal control for luciferase activity. Forty eight hours post-transfection, the cells were lysed and luciferase assays were conducted using the dual luciferase assay system (Promega, Madison, WI, USA). Each experiment was performed in triplicate.