Mir 22 inhibitor

The mimics control with scrambled sequence, miR-106a mimics, miR-106b mimics, miR-124 mimics, inhibitor control with scrambled sequence, miR-106b inhibitor, miR-124 inhibitor, miR-22 inhibitor, and miR-29b inhibitor were purchased from GenePharma (GenePharma Co., Ltd., Shanghai, China). Transfection of miRNA mimics/inhibitor or their corresponding control (20 nM) was performed using the HiPerFect Transfection Reagent (Qiagen) according to the manufacturer’s instruction.

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.