Prl tk

The pNFκB-luc, pSTAT3-TA-luc, and pRL-TK plasmids were purchased from Beyotime. NPC cells were seeded in BeyoGold™ 96-Well White Opaque Plates (Beyotime) transfected with Lipofectamine™ 3000 Reagent (Invitrogen, Carlsbad, CA, USA). Reporter enzyme activity was determined with a dual-luciferase reporter assay system (Beyotime) according to the manufacturer’s instructions. The luminescence signal was determined with a Varioskan LUX multimode microplate reader (Thermo). Relative luminescence units = Firefly luciferase activity/Renilla luciferase activity.

The OFs transfected with pGRE-luc (Beyotime, Shanghai, China) and pRL-TK (Beyotime, Shanghai, China) were incubated with lysate, and 300-μl samples were taken into the chemiluminescence detection tube. 500 μl 1× firefly luciferase reaction solution (Solebo Technology Co., Ltd., China) was added to the reaction tube. The Junior LB 9509 chemiluminescence detector detected the luminescence value for 10 s. 500 μl 1× Aquin luciferase reaction solution (Sobibor Technology Co., Ltd., China) was added and gently blown and mixed. The luminescence value was detected by a Junior LB 9509 chemiluminescence detector for 10 s. Relative luciferase activity was calculated based on the two luminescence obtained in each group.

Genomic DNAs and total RNA were extracted from the hepatopancreas using the DNA Kit (Biomarker, Beijing, China) and Total RNA Isolation Kit (Biomarker) according to the manufacturer's protocol. Specific primers targeting the promoter of Mttp, Apob, and the coding sequence of Hnf4α were designed according to the goldfish reference genome (ASM336829v1; Table 1). PCR products were then cloned into the pEASY-T1 simple cloning vector (Transgen, Beijing, China) and sequenced by Sangon Biotech (Shanghai, China).
The 293T cells (GDC0187) obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China) were maintained in DMEM supplied with 10% fetal bovine serum. Once reached 40%–50% confluence, the cells were seeded into 24-well plates and cotransfected using the Lipo2000 system (Invitrogen, Carlsbad, America) at 70% confluence. For each experiment, 300 ng of pcDNA-HNF4α plasmid, 180 ng of pGL3-Mttp or pGL3-Apob plasmid, and 20 ng of Renilla luciferase reporter plasmid (pRL-TK, Beyotime, China) were mixed and transfected at 26°C. PGL3-Basic and pcDNA3.1 were used as controls in the dual-luciferase reporter assay.

The normal and mutant of the 3'UTR (untranslated region) of the pig VCL gene (Gene ID: 396974) were amplified by PCR and inserted into the pGL3-basic vector (Promega) with Xbal digestion (the sequences refer to Supplementary Data Sheet 1). The PK-15 cells were cultured with Dulbecco's minimum essential medium (DMEM, Gibco) containing 10% fetal bovine serum (Gibco) at 37°C in a humidified 5% CO₂ atmosphere. When the PK15 cells grew to about 75% confluent, ssc-miR-21-5p (100 nM) and a luciferase reporter vector containing the pig VCL-3′-UTR (400 bp) (0.3 μg/well) were co-transfected into the cells using Lipofectamine 3000 (Invitrogen), together with 0.1 μg/well of pRL-TK (Beyotime). At 48 h after transfection, the luciferase activity was measured with a microplate reader (TECAN) according to the manufacturer's instructions.

The wild-type or mutant predictive binding site of miR-2861 on L13Rik was cloned into the PGL3 vector (Fenghui Biotechnology, Hunan, China) to construct pGL3-L13Rik-WT or pGL3-L13Rik-Mut plasmids. The 3′-UTR of CDKN1B or its mutant was inserted into the PGL3 vector to construct pGL3-CDKN1B-3′UTR-WT or pGL3-CDKN1B-3′UTR-Mut plasmids. HEK293 cells (1 × 10⁶) were plated into 24-well plates. pGL3-L13Rik-WT (20 ng), pGL3-L13Rik-Mut (20 ng), pGL3-CDKN1B-3′UTR-WT (20 ng), or pGL3-CDKN1B-3′UTR-Mut (20 ng) plasmids were co-transfected with 40 nM of miR-2861 and 2 ng of pRL-TK (Beyotime, Shanghai, China) into HEK293 cells with Lipofectamine™ 3,000 according to the manufacturer’s protocol. After incubation at 37 °C for 48 h, luciferase activities were assessed using the Dual-Luciferase Reporter Gene Assay Kit (Beyotime, Shanghai, China) in accordance with the manufacturer’s protocol. Renilla luciferase was used for internal control.

For the 3′UTR luciferase reporter assay, the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, WI, USA) was used. The oligonucleotide sequences (wild and mutated type) used are shown in Table S3. The oligonucleotides were ligated into the NheI–XhoI site of pmirGLO. The RAW264.7 cells were co-transfected with the miR-146a mimic (or its control) and constructed pmirGLO vectors and pRL-TK (Promega, WI, USA) using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions for 24 h. For the NF-κB activity assay, RAW264.7 cells were transfected with 1 ug NF-κB luciferase reporter plasmid (Beyotime, Nantong, China), 50 ng pRL-TK, and 50 nM miR-146a mimic or its control for 24 h and then cells were stimulated with 1 µg/ml LPS or H/R. Firefly luciferase and Renilla luciferase luminescence was measured using the Dual-Glo luciferase Reporter Assay System (Promega, WI, USA) and a GloMax 20/20 Luminometer (Promega, WI, USA). Ratios of Firefly luciferase luminescence relative to Renilla luciferase luminescence were calculated.