Si rna β catenin

The 3′-UTR of the β-catenin mRNA CTNNB1 (accession no. NM_001098209.1) was amplified using PCR and subcloned into the pGL3 vector MCS cloning site (Promega Corporation, Madison, WI, USA). CTNNB1 was classified as either wild type (WT) or mutant (Mut, binding sites for miR-214), and pGL3-WT-CTNNB1-3′-UTR and pGL3-Mut-CTNNB1-3′-UTR plasmids were generated. The sequence for the CTNNB1 3′-UTR forward primer was 5′-TCTAGAATACAATGACTT TTTAGCTG-3′ and that for the reverse primer was 5′-TCTAGATTAGCCAAG-3′.
miR-214 mimic (sense, 5′-ACAGCAGGCACAGACAGGCAGU-3′ and antisense, 5′-UGCCUGUCUGUGCCUGCUGUUU-3′), mimic negative control (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′), miR-214 inhibitor (5′-UGCCUGUCUGUGCCUGCUGUUU-3′), inhibitor negative control (5′-CAGUACUUUUGUGUAGUACAA-3′), and small interfering (si)RNA-β-catenin were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). The Dvl1 expression vector pCS2-Myc-Dvl1 was provided by Dr. Zhenge Luo (Institute of Neuroscience, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences). NSCLC cells were transfected with the above plasmids or miRNAs (final concentration: 20 nM) using Lipofectamine 2000 (Thermo Fisher). Transfected cells were incubated for 48 h at 4 °C for further experiments.

The human ESCC cell lines TE-1 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and TE-10, KYSE-150, and ECA-109 cells were a gift from Dr. Ye Hua of the Clinical Medicine College at Jiangsu University. The human esophageal epithelial cell line, Het-1A, was purchased from Jennio Biological Technology (Guangzhou, China). Cells were respectively maintained in RPMI 1640 or DMEM (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (HyClone, Logan, UT, USA) and cultured at 37 °C in 5% CO₂. IL-23, anti-IL-23, and anti-IL-23R were purchased from R&D Systems (Minneapolis, MN, USA), and WHI-P154, DAPT, and ICG-001 were purchased from Selleck Chemicals Inc. (Houston, TX, USA). For RNA interference, cells were transfected with siRNA oligos (Gene Pharma, Shanghai, China) against human β-catenin and Notch1 using Lipofectamine 2000 (Invitrogen, San Diego, CA, USA). The following siRNA sequences (GenePharma, Shanghai, China) were used: siRNA-β-catenin: sense, 5′-GUCCUGUAUGAGUGGGAACTT-3′; antisense, 5′-GUUCCCACUCAUACAGGACTT-3′. siRNA-Notch1: sense, 5′-GGGCUAACAAAGAUAUGCATT-3′; antisense, 5′-UGCAUAUCUUUGUUAGCCCTT-3′. Negative controls using non-transfected cells and empty vector-transfected cells were performed in parallel. The colony formation assay and soft agar assay were described previously [22 (link)].