AIH animal model and miRNA transfection
Eight-week-old male C57BL/6 mice were obtained from Vital River (Beijing, China). AIH in mice was induced by intravenous injection of a single dose of freshly prepared Con A (15 mg/kg, Sigma-Aldrich Chemical Co., China). After Con A administration, blood samples were collected for measurement of plasma alanine transaminase (ALT) and aspartate aminotransferase (AST) levels using mouse AST ELISA kits (Biotron Diagnostics, Hemet, CA, USA), according to the manufacturer’s instructions. MiR30a expression is regulated in vivo by miR30a agomir or antagomir. MiR30a agomir, antagomir, or their negative controls (all form RiboBio, Guangzhou, China) were injected via the tail vein in mice 72 h before Con A administration (n = 5 animals per group).
All experimental animal protocols followed the regulations for the Administration of Affairs Concerning Experimental Animals (AdminReg, China) and were approved by the Ethics Committee of Animal Experiments and monitored by the Department of Experimental Animals of the Third Affiliated Hospital of Sun Yat-Sen University LingNan Hospital. All the authors confirmed that the experiments complied with the ARRIVE guidelines.
TargetScan and luciferase reporter assay
TargetScan 7.0 was used to predict the target genes interacting with miR30a. A list of candidate miR30a targets was obtained. Among them, Klf14 appeared to be a promising target, as it was implicated in the pathology of inflammation in the liver.
To confirm the relationship between miR30a and Klf14, fragments of the 3′-UTR (Wt) containing the binding site of miR30a, or a 3′-UTR mutant (Mut) of Klf14 were cloned into pMIRREPORTTM luciferase vectors (Huayueyang, Beijing, China). HCs were co-transfected with the luciferase reporter vector, Renilla luciferase control vector (pRL-hTK), and the miR30a agomir or negative control using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA). Luciferase assays were performed 48 h after transfection using the dual-luciferase reporter assay system (Promega, San Luis Obispo, CA, USA) according to the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.
Cell isolation and culture
Primary mouse HCs and liver mononuclear cells (MNCs) were isolated as previously described 11. Briefly, mouse livers were perfused with liberase TM solution (Sigma-Aldrich, St. Louis, MO, USA) after euthanasia, filtered through a 70 μm nylon cell strainer (BD Falcon, Franklin Lakes, NJ, USA), and centrifuged at 20× g for 5 min. The pellets were suspended and placed on the surface of 30% Percoll solution, centrifuged at 1000 × g for 10 min at 4 °C and washed once with phosphate-buffered saline (PBS). HCs were used for mRNA/miRNA isolation or protein extraction. Supernatants containing MNCs were collected, resuspended in 30% Percoll, and gently overlaid onto 70% Percoll. After centrifugation at 1000 × g for 30 min, liver MNCs were harvested from the interphase, washed twice with PBS, and then resuspended for further fluorescence-activated cell sorting (FACS) analysis.
RNA isolation and quantitative reverse transcription-polymerase chain reaction
Total miRNAs from HCs were isolated using the mirVana™ miRNA isolation kit (Ambion, Austin, TX, USA) following the manufacturer’s instructions. RNA was isolated from liver tissue after exosome treatment using TRIzol reagent (Life Technologies, MD, USA) and digested with DNase I (Fermentas, Glen Burnie, MD, USA) according to the manufacturer’s protocol. Subsequently, total RNA was reverse-transcribed to cDNA using a RevertAid First Stand cDNA Kit (Thermo Fisher Scientific, Rutherford, NJ, USA). The expression levels of miR30a, Klf14, and inflammatory cytokines were quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using a 7500 Fast Real-Time PCR System (Applied Biosystems, Frederick, MD, USA) and were normalized to the expression of RNU6B (U6B), RNU44, or GAPDH. All samples were prepared and analyzed three times individually. The PCR primers used for the genes of interest are listed in Supplementary Table S1 online.
Western blotting
Total protein was extracted from HCs using Radioimmunoprecipitation buffer. Western blot analysis was performed as previously described 28. Proteins were loaded into gels, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were incubated with anti-Klf14 polyclonal antibody (rabbit anti-mouse, 1:1000, PA5-23784, Invitrogen, Carlsbad, CA, USA), anti-β-actin Polyclonal Antibody (Rabbit anti mouse, 1:4000, #4967, CST, Beverly, MA, USA) with gentle agitation overnight at 4 ˚C. The membranes were washed three times for 5 min each with TBST (containing 0.1% Tween-20) and incubated with horseradish-peroxidase-conjugated anti-Rabbit IgG (Invitrogen) followed by chemiluminescence detection using PierceTM ECL western blotting substrate (Thermo Fisher Scientific, Rutherford, NJ, USA) according to the manufacturer’s protocol. The membranes were subsequently analyzed using “Quantity One” software (Bio-Rad Laboratories, Hercules, CA, USA).
Histopathology
After sacrifice, the livers of individual mice were perfused with ice-cold PBS to remove blood components. Livers were cut into 2 × 4 × 4 mm3 sections, fixed in 4% paraformldehyde, and embedded in paraffin. Subsequently, 5 μm slices were then cut at various depths in the tissue sections, stained with hematoxylin-eosin (H&E) and examined under light microscopy to determine the level of inflammation.
Flow cytometric analysis
Flow cytometry was conducted using a BD FACScalibur device (BD Biosciences, USA) and analyzed with FCS express V3. After washing with Hank’s buffer devoid of Ca2+ and Mg2+, 5 × 105 liver MNCs were blocked using in 1% bovine serum albumin at 4 °C for 30 min. Unfractionated cells were stained with allophycocyanin-conjugated anti-CD4 and PE-conjugated anti-CD25 (eBioscience, San Diego, CA, USA) monoclonal antibodies. Cells were incubated at 4 °C in the dark for 30 min, washed with PBS/1% fetal bovine serum, resuspended, and analyzed by flow cytometry using a Becton Dickinson fluorescent activated cell sorter (FACSCantoII, Becton Dickinson Immunocytochemistry Systems, San Jose, CA, USA). FACS-Diva software was used for analysis. A minimum of 2 × 104 gated events was acquired for each sample.
Statistical analysis
SPSS software (version 20.0) was used for all statistical analyses. Data are expressed as the mean ± standard error. All results presented represent data collected from at least three independent experiments. Results were analyzed by using the Student’s t-test. Statistical significance was set at p < 0.05.