β galactosidase

Plasmids carrying the Renila luciferase gene linked to a fragment of the Prdm16
3′UTR harbouring miR-149-3p putative binding sites were co-transfected
into HEK293T cells (Human Embryonic Kidney, purchased from the Type Culture
Collection of the Chinese Academy of Sciences, Shanghai, China, authenticated by
STR Profiling, no mycoplasma contamination) along with control miRNA or
miR-149-3p mimic (Genepharm, Suzhou, China). A mutant 3′-UTR of Prdm16 was
constructed by mutagenesis of miR-149-3p from AGGGAGG into
GGAGGGA. HEK 293T cells were cultured in DMEM (Gibco,
Carlsbad, CA, USA) containing 10% FBS and seeded in 12-well plates. At
24 h after plating, 0.2 μg of firefly luciferase reporter
plasmid, 0.2 μg of β-galactosidase (cat# 10586-014)
expression vector (Ambion, Carlsbad, CA, USA), and equal amounts
(20 pmol) of miR-149-3p mimic or scrambled negative control RNA were
transfected into cells with Lipofectamine 2000 (cat# 11668-019) (Invitrogen,
Carlsbad, CA, USA) according to manufacturer's instructions. A
β-galactosidase vector was used as a transfection control. At 24 h
post-transfection, the cells were analysed using a luciferase assay kit
(cat# E4550) (Promega, Madison, WI, USA). All Experiments were performed in
triplicate wells for each condition and repeated five times independently.

A luciferase reporter assay was performed to check if miR-125b-5p could bind to CREB1. Sequences binding to miR-125b-5p on CREB1 3'UTR were deleted to obtain the mutant. The CREB1 3′UTR wild-type and mutant sequences were inserted into the p-MIR-reporter (Ambion, Austin). The wild-types and mutant sequences were confirmed by sequencing. Subsequently, in 24-well plates, the 293T cells were seeded. At 70% confluency, the same amount of β-galactosidase (Ambion) and luciferase firefly reporter plasmids were transfected, with an equal amount of miR-125b-5p mimic/control mimic. Following a previously reported protocol, we performed luciferase assays [18 (link)].