Mir 149 5p mimic

Normal bladder epithelial cells (HCV-29) and four bladder cancer cell lines (J82, 5637, SW780, and T24) were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). Cells were grown routinely in RPMI-1640 medium (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (Gibco, CA, USA) and cultured in a 37 °C humidified atmosphere containing 5% CO₂. circRNA_100146 over-expression (circRNA) or knockdown (shRNA#1, shRNA#2, shRNA#3) plasmids, miR-149-5p mimic, RNF2 overexpression (RNF2) plasmid, and their control vector were obtained from GenePharma (Shanghai, China). J82 and T24 cells were seeded in 6-well plates 24 h prior to circRNA_100146 or miR-149-5p mimic or RNF2 transfection. When the cells were grown–70-80% confluence, transfection was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

For overexpression, miR-149-5p mimic and miR-NC mimic were purchased from GenePharma (Shanghai, China); the sequence of HOTAIR and coding domain sequence of DCLK1 were cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), respectively. For knockdown, miR-149-5p inhibitor (in-miR-149-5p), siRNA against HOTAIR (si-HOTAIR), and their controls were obtained from GenePharma. Cell transfection was carried out with Lipofectamine™ 2000 (Invitrogen) according to the manufacturers’ instruction. After transfection for 24 h, A549/DDP and H1299/DDP cells were collected for further experiments. Sequences of siRNAs were as follows: si-HOTAIR: 5ʹ- GAACGGGAGUACAGAGAGAUU-3ʹ; si-NC: 5ʹ-GAACGGAGCGAGCAGACCUUU-3ʹ.