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CRISPR-mediated BMP9 ablation promotes liver steatosis via the down-regulation of PPARα expression

Hepatic cytokine BMP9 attenuates fatty liver by regulating the key lipid metabolism factor PPARα.


RNA extraction and real-time PCR
Cellular and total tissue RNA were extracted using TRIzol reagent (Invitrogen, 15596026) according to the manufacturer's instructions. cDNA was synthesized using a PrimeScript TM RT Reagent kit (Takara, RR037A) and PowerUP TM SYBR Green Master Mix (Thermo Fisher Scientific, 1708040) with the following cycling conditions: 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. Real-time PCR was performed using the QuantStudio TM 6 Flex Real-Time PCR System (Applied Biosystems, 4484642). 18s mRNA was used as the control. Relative changes in mRNA expression were calculated using the comparative cycle method (2 −ΔΔCt ). Real-time PCR Primers (Table  S2) were obtained from the online primer database PrimerBank (https://pga.mgh.harvard.edu/primerbank/).

Gene microarray analysis
Three independent 16-week-old littermate WT and BMP9-KO mouse livers were collected and frozen in liquid nitrogen for microarray analysis. The Agilent SurePrint G3 Mouse GE V2.0 Microarray (8*60K, Design ID: 074809) was used in this experiment. Total RNA was quantified by a NanoDrop ND-2000 (Thermo Fisher Scientific), and the RNA integrity was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies). Sample labeling, microarray hybridization and washing were performed based on the manufacturer's standard protocols. Briefly, total RNA was transcribed to double-stranded cDNA, then synthesized into cRNA and labeled with Cyanine-3-CTP. The labeled cRNAs were hybridized onto the microarray. After being washed, the arrays were scanned by an Agilent Scanner G2505C (Agilent Technologies). Feature Extraction Software (version 10.7.1.1, Agilent Technologies) was used to analyze array images to obtain raw data. Genespring (version 13.1, Agilent Technologies) was used to finish the basic analysis of the raw data. First, the raw data were normalized with the quantile algorithm. The probes with 100% of the values in any 1 out of all conditions flagged as "Detected" were chosen for further data analysis. Differentially expressed genes were then identified through the fold change (FC) and the P value calculated by t-test. The threshold for up-and downregulated genes was an FC ≥ 2.0 and a P value ≤ 0.05. Subsequently, GO and KEGG analyses were used to determine the roles of the differentially expressed mRNAs. Finally, hierarchical clustering was performed to display the distinguishable gene expression patterns among the samples.

Promoter dual-luciferase reporter assay
First, the PPARα promoter sequence (1800 bp) was inserted into the pGL3 basic vector (Addgene, 64784), which contains firefly luciferase. The pRL-TK vector (Addgene, 11313) containing renilla luciferase was used as a control. Hepa 1-6 cells were seeded at a density of 10,000 cells per well in flat-bottomed 24-well plates. The cells were cultured in DMEM supplemented with 10% FBS, maintained at 37°C and transfected with 2 µg pGL3 basic vector and 0.1 µg pRL-TK vector using Lipofectamine 2000 reagent (Invitrogen, 11668019). 24 h after transfection, the medium was replaced with 200 µl of fresh DMEM. Then, the Hepa 1-6 cells were treated with (0, 2, 10, or 40 ng/ml) recombinant BMP9 protein for 24 h. The relative luciferase activity was determined using a Dual-Luciferase® Reporter Assay System (Promega, E1910) according to the manufacturer's instructions. Signals were measured using a Microplate System (Promega, GloMax E5311). For further PPARα antagonist treatment, GW6471 (MCE, HY-15372) was dissolved in DMSO and kept at 10 mM. Where necessary, Hepa 1-6 cells were treated with 50 µM GW6471.

ChIP analysis
PPARα promoter was divided into six 300 bp regions. Furthermore, we investigated the precise TF binding sites of the PPARα promoter. We utilized an online promoter prediction tool (http://www.fruitfly.org/cgi-bin/seq_tools/promoter.pl) to generate 3 potential PPARα promoter TF binding sites per 50 bp. Promoter predictions for 1 eukaryotic sequence with score cutoff 0.80. ChIP assays were performed using a Chromatin Immunoprecipitation kit (Merck Millipore, 17-371) according to the manufacturer's instructions. Hepa 1-6 cells were treated with 0 or 10 ng/ml recombinant BMP9 protein for 24 h. After treatment, the cells were lysed by ultrasonic processing using an Ultrasonic Cell Crusher (SONICS Uibra Cell, VCX130). Thereafter, a smad 1/5 Antibody Sampler kit (Cell Signaling Technology, 12656) antibody was added to the cell lysate for immunoprecipitation. The immunoprecipitation products were collected for further semi-quantitative PCR followed by 2% agarose gel electrophoresis analysis and qRT-PCR. Primers for the ChIP assay were designed by the online primer tool Primer3Plus (http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi) (Table S3).

Histology staining
Mouse liver tissue samples were fixed in 5% formaldehyde solution overnight and then embedded in paraffin. The tissue sections were stained with hematoxylin and eosin (H&E), Oil Red O, Masson, Sirius Red for the evaluation of the tissue morphology, according to standardized protocols. Cellular lipid droplets were visualized by an Oil Red O Staining kit (Servicebio, G1015L).

Metabolic cage studies to calculate energy expenditure in mice
Based on their home cages, mice were housed in a monitoring system that measures physical activity, food intake, oxygen and carbon dioxide concentrations. Sample data were collected at 14 min intervals for a 4-day period with light (7 a.m.) and dark (7 p.m.) cycle every 24 hours. Oxygen consumption and carbon dioxide production are exported as ml per kg lean mass per h by using an adjusted mean lean mass.

Nuclear magnetic resonance (NMR) analysis
Mouse fat and lean mass were calculated by time-domain-NMR (TD-NMR) by using a Minispec Live Mouse Analyzer (TESTNIUMAG, MesoMR23-060H-I). The littermate BMP9 WT and BMP9 KO mice were anesthetized and scanned with an NMR system. After scanning, the proportion of muscle and fat and the distribution of fat in mice were obtained.

ELISA assay
The clinical blood samples were incubated at room temperature for 4 h to separate the serum. Human serum BMP9 levels were detected by using a Human BMP9 ELISA kit (RayBiotech, ELH-BMP9). Similarly, the blood was taken from the eyes of mice, and the mouse serum was isolated by centrifugation. Mouse serum BMP9 levels were detected using a Mouse BMP9 ELISA kit (RayBiotech, ELM-BMP9) and Mouse FGF-21 ELISA Kit (Abcam, ab212160). For all operating procedures, refer to the instructions provided by the manufacturer.

BMP9-overexpressing AAV construction
An AAV8 vector named GPAAV-WPRE (Genomeditech, Shanghai), was used in this study. A mouse BMP9 coding sequence was inserted into this AAV8 vector at Bam HI-EcoR I site. AAV-Vector and AAV-BMP9 vectors were transfected into 293T cells (Cobioer, CBP60439) with Lipofectamine 2000 reagent (Invitrogen, 11668019). The viral supernatants were collected as previously described. Viral genome titers were determined by quantitative PCR.

Administration of the BMP9 overexpression AAV in mice
16-week-old BMP9-KO mice were anesthetized with an intraperitoneal injection of chloral narcotic (Servicebio, WG). Thereafter, the concentrated AAV-Vector and AAV-BMP9 viruses with established titers were diluted in 0.9% sodium chloride injection buffer (Baxter, A6E1323) and injected via the tail vein at 2 × 10 12 vg per kg mouse. For short term treatment, the GTT and ITT were performed on these AAV-treated mice after 2 months injection. Whereafter, mice were sacrificed. The blood, livers and other tissues were collected. For long term (4 months) treatment, the changes in mouse body weight were continuously monitored after the injection of AAV.