Hek293t cells

The MKNK2 3′‐UTR containing either the seed sequence of wild type or mutant miR‐223‐3p binding site was inserted into the pmirGLO vector (Promega, USA). HEK293T cells (purchased from ATCC, USA) were seeded at 1 × 10⁶ cells/well in 24‐well plates were transfected with the pmirGLO‐MKNK2‐Wt/Mut and miR‐223‐3p mimics or scrambled miRNA (synthesized by GenePharma) using Lipofectamine 2000 (Invitrogen). After 48 h, the cells were lysed for measurement with a luciferase reporter assay kit (Promega).

Based on bioinformatics, we predicted a binding site between miR-335 and VapB. RiboBio Biology (Guangzhou, China) was entrusted to amplify the complementary binding sequence and mutation sequence of miR-335 with VapB and cloned it into vectors to construct WT and mutant plasmids VapB-WT and VAPB mutated type. According to the instructions of Lipofectamine 2000, the plasmids were co-transfected with mimic NC or mimic miR-335 (GenePharma) respectively into HEK293T cells (Shanghai Institute of Cell Biochemistry, Chinese Academy of Sciences). The binding relationship between miR-335 and VapB was verified using Dual-Glo^® dual-luciferase reporter gene detection system (E2920, Promega Corporation, Madison, WI, USA)^{26 (link)}.

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.