2.1. Hepatic HNF4α expression in NAFLD patients
Hepatic HNF4α expression were compared between thirty three NAFLD patients and sixteen healthy control. NAFLD was defined as the case of more than 5% of hepatocytes in which fat deposition was observed in liver tissue. And viral hepatitis A, B, and C, drug-induced hepatitis, alcoholic and/or autoimmune liver disease was excluded. Patients with alcohol consumption of > 210 g/week for men and > 140 g/week for women were also excluded from NAFLD. For control group, normal liver tissue was obtained from 16 patients through hepatic resection because of benign disease. All control subjects had normal liver enzymes and < 5 % hepatic fat content.
Informed consent was waived from institutional review board (IRB) at Hanyang University Hospital.
Human tissue samples performed in accordance with IRB of Hanyang University Hospital guidelines and The study was approved by the IRB of Hanyang University Hospital.
The protocol was registered at the Clinical Research Information Service (
http://cris.nih.go.kr/cris/index.jsp) with the registration number KCT0000900.
2.2. Hepatic HNF4α expression NAFLD animal models
Hepatic HNF4α expression was evaluated in two types of animal NAFLD models [24 weeks high fat (HF) and 14 weeks methionine and choline deficiency (MCD) diets models].
Animal study performed in accordance with ARRIVE guidelines and The study was approved by by the Hanyang Institutional Animal Care and Use Committee (IACUC) (HY-IACUC-11-067).
All methods were carried out in accordance with ARRIVE guideline and regulations of IACUC at Hanyang University.
2.3. Histology assessment
To assess hepatic steatosis, liver tissue sections were scored for activity (degree of inflammation) and stage (degree of fibrosis) of disease according to the histological grading and staging systems, respectively. Hepatic steatosis was graded as follows: < 5 % (score, 0); 5 %–33 % (score, 1), > 33 %–66 % (score, 2) and > 66 % (score, 3); steatosis, 0–3; lobular inflammation, 0–4; portoperiportal activity, 0–4; and fibrosis, 0–4. For NAFLD, the histological changes were graded using the NASH clinical research network scoring system 13. A single blinded pathologist evaluated histological characteristics and calculated overall steatohepatitis scores.
2.4. Immunohistochemistry
Formaldehyde (10%) fixed and paraffin-embedded liver tissue sections were stained with hematoxylin and eosin. Four-micrometer thick sections were cut from paraffin block and coated on a glass slide. Immunohistochemistry for HNF4α, NTCP and, Cyp7a1 (Abcam, Cambridge, UK), expressions (Abcam, Cambridge, UK) was performed. A single blinded pathologist evaluated histological characteristics, intensity, extent and immune-reactive socres of immune-stained sections.
2.5. shHNF4α construction and animal study
Ad-shHNF4α and Ad-empty (control) have been constructed. When Ad-HNF4α was constructed, only the HNF4α coding region (which does not include any 3′UTR) was cloned into the adenoviral vector. Ad-shHNF4α and Ad-empty (control) were generated by cloning the HNF4α coding region into a pAdEasy-1 vector (catalog #240005, Agilent), followed by transfection into HEK 293 cells for adenovirus production. Cells were infected with adenoviruses at an MOI of 10. Mice were intravenously (via tail vein) injected with 2.5 ×106 pfu adenoviruses. The viral titers (plaque-forming units) were determined in HEK293 cells using standard procedures. Comparable amounts of Ad-RGD were used as a control.
The use of animals in this work was in accordance with the Hanyang Institutional Animal Care and Use Committee.
Forty-five male C57BL/6 mice were randomly divided into the following groups: Control (n = 15) fed on MCD diet, and Sh-HNF4α (n = 15) fed on MCD diet with Ad-shHNF4α injection for 14 weeks. Animals were maintained in a temperature-controlled room (22 ∘C) on a 12:12 h light-dark cycle. Tail vein injection was performed using a 1mL insulin syringe with 28 gauge. Mice were given daily tail vein injections with saline, control shRNA, or shHNF4α for 4weeks from 10th week to 14th week. Mice were restrained for a single 300µl tail vein injection of either saline, 2.5 x 10
12 viral particles of adenovirus expressing nontargeting control shCon or 2.5 x 10
12 viral particles expressing shRNA targeting HNF4α. Body weights of mice were assessed weekly. Four-week post-injection, animals were humanely euthanized by CO2 inhalation and plasma and liver samples were collected for analysis.
2.6. HNF4α overexpression in In Vitro system
The coding regions of the human HNF4α gene from TrueORF cDNA Clones (Origene, Rockville, MD) was amplified using PCR. The fragment was cloned into pECFP (enhanced green fluorescent protein)-C1 vector (Clontech, Palo Alto, CA). To generate the HNF4α construct, PCR products were subcloned into pGEM-T easy vector (Promega, Madison, WI), and then cloned into EcoRI - BamHI sites of the pECFP-C1 vector. For HNF4α overexpression, electroporation was performed with Microporator (Thermo Fisher Scientific, Waltham, MA USA) using the manufacturer’s recommended settings (1400voltage, 20ms, two pulses). For quantitative measurement of transfection efficiency, the percentage of transfected cells was measured using LightCycler 480 system® (Roche Diagnostics, Mannheim, Germany) after electroporation.
2.7. siRNA knockdown
For HNF4α knockdown, HepG2 cells were transfected with Silencer predesigned human HNF4α siRNA and Negative Control #1 siRNA (Thermo Scientific, Waltham, MA, USA) using Lipofectamine RNAi/MAX transfection reagent (Invitrogen) following the manufacturer’s instructions. Two days after siRNA transfection, cells were collected for analysis.
2.8. Cell culture
HepG2 cells obtained from American Type Culture Collection (Rockville, MD) were grown in Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 10 % fetal bovine serum and penicillin (50 U/ml)/streptomycin (50µg/ml) (Invitrogen).
2.9. Western blot analysis
Liver tissue samples were solubilized in radioimmunoprecipitation assay buffer containing protease inhibitors (Pierce, Rockford, IL). The lysates were centrifuged (13,000 g for 10 min at 4°C) and supernatants were boiled with sodium dodecyl sulfate buffer (0.5 M β-mercaptoethanol). Later, the lysates were subjected to 12% SDS-PAGE for western blot analysis. Reactive protein bands were analyzed by enhanced chemiluminescence detection reagent (Amersham Biosciences, Piscataway, NJ) using an image reader LAS-3000 (version 2.1; Fujifilm, Tokyo). HNF4α polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted 1:500 in TBS-T/5 % BSA. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody diluted 1:2000 (Santa Cruz Biotechnology, Inc.) in TBS-T/5 % BSA was used as a secondary antibody. The experiment was repeated using three different samples.
2.10. TUNEL assay
The TUNEL reaction was carried out using the "In situ cell death detection kit, fluorescein" (Roche, USA) according to the manufacture's instruction. Confocal imaging was performed using a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).
2.11. MTT assay
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was purchased from Sigma-Aldrich (Saint Louis, MO, USA). HepG2 cells were seeded onto 96-well plates (5,000 cells/well) using DMEM. After 24h, cells were incubated with palmitic acid (PA; 400µM), chenodeoxycholic acid (CDCA;100µM), a combination of CDCA and PA, and DMSO (mock control) for 24h. After treatment incubation, MTT solution (0.25mg/mL MTT in PBS, pH 7.4) was added to each well followed by incubation at 37°C for 4h. The supernatants were removed from wells and DMSO was added to dissolve crystals. The optical density was measured at 540nm using an ELISA microplate reader (Tecan, Research Triangle Park, NC).
2.12. Statistical analysis.
All quantitative data were expressed as group means and standard deviations. Statistical analysis was performed with by Kruskal-Wallis test and Mann Whitney’s U-test using SPSS for Windows version 18.0 (SPSS Inc., Chicago, IL, USA). p values less than < 0.05 were considered significant.