Mir 22 mimic

Histone H3 monoclonal antibody, phospho-I-κB monoclonal antibody, and NF-κB p65 (L8F6) mouse monoclonal antibody were purchased from Cell Signaling Technology (US). β-actin monoclonal antibody was purchased from Santa Cruz (US). Horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Pierce (US). Dylight594-conjugated secondary antibody was purchased from Genshare Biological (China). IPEC-J2 cell line was kindly gifted by Dr. Zhanyong Wei (Henan Agricultural University, China). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F-12/HAM (Thermo Fisher Scientific, US) supplemented with 100 IU of penicillin and 100 mg of streptomycin per ml, at 37 °C in an incubator with 5% CO₂. The TGEV Shaanxi strain was separated from TGEV-infected piglets [47 ]. miR-22 mimics, miRNA mimics control, miR-22 inhibitors, miRNA inhibitors control, siCirc009380, and negative control were synthesized by GenePharma (China) (The sequences were shown in Additional file 1: Table S8).

3' UTRs of candidate target genes containing the binding site of miR-22 were respectively amplified by PCR and were cloned into the vector psiCHECK-2 (Promega, US). To obtain mutation of miR-22 complementary sites within the 3' UTR of HK2 and IL-6, the binding sites of miR-22 seed region in 3' UTRs were mutated following a mutagenesis protocol 29 (link) (The primers are shown in Table S4). The miR-22 mimics, miR-22 mimics control, miR-22 inhibitor, and miR-22 inhibitor control were designed and synthesized by Genepharma (Shanghai, CHN) (The sequences are shown in Table S3). For the luciferase reporter assay, IPEC-J2 cells were seeded in 24-well plate and then co-transfected with 100 ng plasmid and 100 nM of miR-22 mimics, or miR-22 inhibitor, or control, using Lipofectamine 3000 (Invitrogen, US) according to the manufacturer's instructions. At 48 h post transfection (hpt), the luciferase activities were measured using Dual-Glo Luciferase Assay System (Promega, US) following the manufacturer's manual.