Let 7d mimic

A 3′ untranslated region (3′UTR) fragment (1078-bp) of the HMGA2 containing the let-7d binding region was synthesized and purified by Generay biotechnology (Shanghai, China). Then the fragment was ligated into pGL3 dual luciferase vector (Promega, Madison, USA). IK cells at a density 5 × 10⁵ cells per well were co-transfected with 1 μg of pGL3-HMGA2 vector and 40 pmol of let-7d mimic or antagomir (Ribobio, Guangzhou, China) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Transfection of the negative control of mimic and antagomir was also performed as a control. After 24 h, the luciferase activities of firefly and Renilla were continuously measured by a Dual Luciferase Reporter Assay kit (Vazyme). The firefly luciferase activities were normalized to the Renilla luciferase activities to calculate the relative luciferase activity of HMGA2. The sequence of let-7d mimic, antagomir and their negative control we used are as following:

miR-NC: 5′-UCACAACCUCCUAGAAAGAGUAGA-3′;

let-7d mimic: 5′-AGAGGUAGUAGGUUGCAUAGUU-3′;

antagomir-NC: 5′-UUUGUACUACACAAAAGUACUG-3′;

let-7d antagomir: 5′-AACUAUGCAACCUACUACCUCU-3′.

For transfection, cells were first washed once with serum-free medium and then incubated in 4 mL of serum-free medium for 4-6 h. miRNA mimic (100 nM) or other constructs (1 µg/mL for PFL and shRNA-PFL) and lipofectamine 2000 (Invitrogen, Carlsbad, CA) were separately mixed with 500 μL of Opti-MEM ^® I Reduced Serum Medium (Gibco, Grand Island, NY) for 5 min. Then, the two mixtures were combined and incubated at room temperature for 20 min. The lipofectamine:miRNA (or plasmid) mixture was added to the cells and incubated in 6-well culture plates at 37°C for 6 h. Let-7d mimic was purchased from RiboBio Co., Ltd. (Guangzhou, China). Subsequently, 5 mL of fresh medium containing 10% FBS was added to the flasks and the cells were maintained in the culture medium for 48 h until the subsequent experiments.