Mir 145 inhibitor

Short hairpin RNAs (shRNAs/sh) targeting MEG3 (forward, 5′-CCGGATAGAGGAGGTGATCAGCAAACTCGAGTTTGCTGATCACCTCCTCTATTTTTTG-3′ and reverse, 5′-AATTCAAAAAATAGAGGAGGTGATCAGCAAACTCGAGTTTGCTGATCACCTCCTCTAT-3′), miR-145 mimic (forward, 5′-GUCCAGUUUUCCCAGGAAUCCCU-3′ and reverse, 5′-AGGGAUUCCUGGGAAAACUGGAC-3′) and negative control (NC)-mimic (forward, 5′-UCACAACCUCCUAGAAAGAGUAGA-3′ and reverse, 5′-UCUACUCUUUCUAGGAGGUUGUGA-3′), miR-145 inhibitor (5′-AGGGAUUCCUGGGAAAACUGGAC-3′) and NC-inhibitor (5′-UCUACUCUUUCUAGGAGGUUGUGA-3′) were obtained from Shanghai GenePharma Co. Ltd. An empty plasmid cloning (pc)DNA3.1(+) vector (Addgene, Inc.) was used as a control for MEG3 and PPARγ overexpression pcDNA3.1(+) constructs. LX-2 cells (5×10⁵ cells/well) were seeded into 6-well plates for 24 h, and then transfected with the aforementioned recombinant vectors (2 µg), shRNA (1 µg), miR-145 mimic (20 nM), miR-145 inhibitor (20 nM) or the controls, at 40–60% confluence using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Transfected cells were harvested 48 h after transfection.