Cell culture
Knockdown of TM6SF2 gene was mediated by the lentiviral vector pLenti6.3-MCS-TM6SF2-EGFP (sh-TM6SF2) or pLenti6.3-MCS-EGFP (sh-Ctrl). The target sequences for sh-TM6SF2: TGACCTGGCCCTTGTCATATA. After two or three generations of antibiotic screening, the expression of TM6SF2 was evaluated through immunoblotting. All cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Cat. No: C125C5, NCM Biotech, Suzhou, China) at a 37˚C, 5% CO2 condition. Cells were cultured under 1% O2 and 5% CO2 condition for 24 h to simulate a hypoxia condition. As for starvation condition, cells were cultured in DMEM containing 0.1% FBS for 24 h.
Western blot
Cells in a 6-cm dish with 80% confluence were lysed with 250 ul of RIPA Lysis Buffer, centrifuged at 13000 rpm. The quantification of supernatant collected was assessed by BCA method (Beyotime, Jiangsu, Chang). After electrophoresis and proteins was transferred to a 0.45μm PVDF membrane according to the standard immunoblotting protocol. The membrane was blocked in 5% fat-free milk and then maintained in primary antibodies for 12 h at 4℃. After washing, the membrane was incubated with HRP-labeled goat anti-rabbit (mouse) antibody at 25℃ for 60 min and visualized with ECL reagent (Millipore, MA, USA). The supplementary material listed antibodies used in this study.
Clinical Specimens
Liver specimens were collected from Shanghai General Hospital. When 5% or more of hepatocytes had steatosis, the diagnosis of NAFLD was confirmed. This study was approved by the Ethics Committee of Shanghai General Hospital and proceeded strictly in accordance with the declaration of Helsinki. All patients or their family members have been fully informed and signed the written consents.
Immunohistochemistry.
The paraffin-embedded liver tissue was deparaffinized with standard protocols and rehydrated. After antigen retrieval, the tissue was blocked and incubated with the anti-TM6SF2 (1:200) antibody overnight at 4℃. Then the slides were washed and incubated with biotin-labeled goat anti-mouse IgG (H+L) at 37℃ for 15mins and developed with DAB work solution. Images were obtained by a Leica microscope (Germany).
Real-time RT-PCR assay
A total of 50 mg of liver samples from mice or human was lysed with RNAiso Plus reagent (Takara Biotechnology, Otsu, Japan) and total RNA were extracted according to the standard protocol. The reverse transcription of 500ng RNA was performed using the RNA PCR Kit (Takara Biotechnology) and the resulting cDNA was used as the PCR template. Quantification of target gene expressions was assessed by LightCycler®96 (Roche, Switzerland) using a Hieff® qPCR SYBR Green Master Mix (No Rox) kit (Yeasen Biotechnology Co., Ltd, China). The mRNA level of β-actin was considered as the endogenous control. The primers used were listed in Supplementary material.
Experimental animals
The AAV system (type 8) expressing TM6SF2 shRNA (AAV-shTm6sf2) was employed to regulate the levels of TM6SF2 in C57BL/6J mice (male). The corresponding controls are AAV-shNC. All mice were firstly under a normal chow diet (NCD) condition for 3 days, and then 100 ul of AAV8 virus (2×1011) was injected into the tail vein. The transfection efficiency was determined by immunoblotting. All mice weighing 19-22 g (aged 4-6 weeks) were housed in a 23±2°C environment with a 12h light/dark cycle. All mice were allowed water ad libitum and fed a high-fat diet (HFD) or NCD continuously for 16 weeks. As for MK-4074 treatment, mice were primarily fed an 8-week HFD for inducing NAFLD phenotypes and then were administered orally with MK4074 (10mg/kg/day) or placebo (normal saline) for additional 8 weeks on the same diets.
Evaluation of serum parameters and hepatic lipid content
The concentrations of cytokines, metabolites, and hepatic enzymes in serum were determined using commercial kits (ab208348 for TNF, ab197742 for IL-1β, ab222503 for IL-6, JLC049 for CCL2, JLC5800 for CXCL10, ab180875 for acetoacetate, ab180876 for β-hydroxybutyrate, C010-2-1 for AST and C009-2-1 for ALT) according to the manufacturer’s instructions. The hepatic lipid content and malonyl-CoA levels were also measured using commercial kits (A110-1-1 for TG, A111-1-1 for TC, A042-2-1 for NEFA, JL47416 for malonyl-CoA) and normalized by the total protein. Details of all commercial kits used were listed in Supplementary material.
Microarrays
After palmitic acid (PA, 150 μM) stimulation for 24 h, total RNA was extracted from both TM6SF2-knockdown L02 cells and corresponding controls using RNAiso Plus (Takara Biotechnology). Microarray gene expression analysis was performed by illumina sequencing. We analyzed differentially expressed genes (DEGs) between sh-Ctrl and sh-TM6SF2 cells by using the “edgeR” package [21]. Fold change >1.2 and adjusted P value < 0.05 were set as the screening cutoffs for the upregulated DEGs. These DEGs were further analyzed via DAVID 6.8 (https://david.ncifcrf.gov/) for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The “ggplot2” package in R is used for result visualization.
Preparation of fatty acid solution
A 5 mM stock solution of palmitic acid (PA, Sigma-Aldrich) was obtained by dissolving PA in 3% of bovine serum albumin (BSA, Sigma-Aldrich) medium by continuous stirring for ~4 h with a 60 °C water bath. The stock solution was then diluted by DMEM medium to achieve the designated concentration and 3% of BSA was used as a control. The concentrations of free fatty acid had been reported to range between 0.1 and 0.7 mM [22-24].
Glucose and insulin tolerance test
Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed as previously described [25]. After the above steps were finished, the glucose concentrations of blood collected from the tail vain at indicated points were analyzed immediately by a glucose meter (Yuwell, China).
Nile red staining
After sh-ctrl and sh-TM6SF2 L02 cells were constructed, cells were incubated in DMEM medium contained 150 μM of palmitate acid (PA) for 24 h to generate a steatosis cell model. Fixed with 4% paraformaldehyde, cells were then incubated with 0.1μg/ml Nile Red for 15 min. After being washed with PBS, cells were incubated with DAPI (Solarbio) for additional 5 min. Images were taken with a Leica microscope (Leica, Germany) and quantified by Image J software.
Magnetic Resonance Imaging (MRI)
MRI imaging were performed on the 7.0 T Bruker BioSpec MRI scanner (Bruker, USA). Before imaging, mice fasted overnight to avoid the disturbance of stomach contents on liver imaging. The mice were first anesthetized with oxygen contained 2-3% isoflurane, and then placed in a prone position with their heads facing inward. Throughout the imaging process, mice were continuously provided with oxygen mixed with 1-3% isoflurane through the nosecone to maintain the respiratory rate at 50-70 breaths/minute. All mice underwent abdominal MRI to acquire the maximum cross-sectional area of liver. The region of interest was analyzed by Image J software, and the size of the cross-section area is represented by number of pixels.
Histological analysis
Liver sections were embedded in paraffin followed by staining with haematoxylin and eosin (H&E) and frozen liver sections were stained with Oil Red O (O8010-5g; Solarbio) for lipid visualization. Images were procured using a light microscope (Leica, Germany). Oil red O- stained sections are quantified by Image J software.
FAO measurement.
A total of 6,000 cells were seeded in a XF96 cell culture micro plate and each group was assayed in 5 or 6 repeated wells. After cell adherence (about 8 h), the growth medium was changed to substrate-limited growth medium (0.5 mM of glucose, 1 mM of GlutaMAX, 0.5 mM of XF L-carnitine, 1% FBS, pH 7.4) for overnight incubation. Before the Seahorse Bioscience XF96 instrument (Agilent, USA) was started, the media was again change to palmitic acid measurement medium. Injectors added different inhibitors (oligomycin 2 μM, FCCP 2 μM and R/A 1.2 μM) to each chamber when the instrument began to measure the oxygen-change rate. The difference between the rate of oxygen consumption in each group revealed the level of fatty acid oxidation (FAO) based on palmitate substrates and the basal respiration, maximal respiration, spare respiratory capacity and ATP production were calculated.
Cell viability Assay
Cell viability was assessed by a CCK-8 (NCM Biotech, China). Sh-ctrl and sh-TM6SF2 cells were seeded in a 96-well plate at a density of 5,000 cells per well and the cell viability assay was performed after PA or fatty acid-free BSA treatment for 24 h. The absorbance (450 nm) was evaluated by the microplate reader (BioTek, USA). Each sample was assayed in 5 repeated wells and the experiment was performed three times independently.
Quantitation of intracellular TG levels in cell lines
After PA or BSA treatment for 24 h, cells were lysed on ice with RIPA buffer (Beyotime, Beijing, China) for 20 min and centrifuged at 13000 rpm for 20 min at 4°C. And then the supernatant was transferred to a new tube. TG (triglyceride) levels were evaluated by a TG detection kit (Nanjing Jiancheng, Jiangsu, China) and normalized by the total protein. The experiment was performed three times independently.
Bioinformatic analyses
Expression profiles of TM6SF2 gene in NAFLD cases retrieved from the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo) database. The accession numbers were GSE130970, GSE48452, GSE83452 and GSE89632.
Statistical analysis
All data were presented as means ± SD. Student’s t-test was used to evaluate the difference between two groups. Differences among more than two groups were compared with one-way ANOVA followed by Tukey’s multiple comparisons test. In vitro studies, all experiments were independently performed three times. SPSS software (version 25.0) was applied in all statistical analyses. P< 0.05 was considered statistically significant and denoted as * P<0.05, ** P<0.01, and *** P<0.001, n.s., not significant.