Animals, diet, and hepatic IRI model
C57BL/6 wild-type (WT) mice were purchased from Shanghai SLAC Co. Ltd (Shanghai, China). Animal protocols were approved by the Institutional Animal Care and Use Committee of Renji hospital, School of Medicine, the Shanghai Jiao Tong University. 4- to 6-week-old male mice (14-16g) C57BL6 mice were fed a high-fat diet (HFD; 18.1% protein, 61.6% fat and 20.3% carbohydrates; D12492, Research Diets, New Brunswick, NJ) for 24 weeks to build a mouse model of liver steatosis. Mice were housed in a standard environment at 22 to 24℃ with a 12:12 h light-dark cycle and ad libitum access to food and water. Successfully established HFD mice received liver IRI treatment as published [30]. In short, arterial and portal venous blood supply to the cephalad lobes of the liver were interrupted with an atraumatic clip for 90 min, and mice were sacrificed after reperfusion for 6h.
Histology
Liver tissue sections were stained with hematoxylin-eosin (H&E) according to standard procedures. Oil red O (ORO) (O0625; Sigma-Aldrich, St Louis, MO) staining were performed following previously described protocol [31].
Enzyme-linked immunosorbent assay (ELISA)
ELISA kits were used to detect mouse IL-6 and TNF-α (NeoBioscience Technology, Shenzhen, China) levels following the manufacturer’s instructions.
Biochemical analysis
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected by microplate test kits (Nanjing Jianchen Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions [32].
RNA-seq
Total RNA was exacted from separated liver tissue using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Then nanoDrop 2000 (Thermo Fisher, Waltham, MA) were applied for the quality and concentration of the isolated RNA. Before circRNA sequencing, we used RNase R (Geneseed, Guangzhou, China) to remove the linear RNAs from total RNA. We contrasted cDNA library with one microgram of the RNA by VAHTSTM mRNA-seq v2 library Prep Kit for illumina (Vazyme, Nanjing, China). Next, the fragmented RNA and double-stranded cDNA was synthesized. In addition, we performed ligation of cDNA fragments to the adapter for end repair and A-addition as reported before. Then the ligated cDNA was amplified by PCR. Illumina Hiseq 2500 was employed for RNA-sequencing [33]. Quantile normalization and further data analysis were conducted by R software. The differentially expressed circular RNAs were selected according to a standard of fold-change and p value (p-value < 0.05 and fold change > 2.0).
Validation of circRNAs
We designed divergent and convergent primers to subsequent analyze these circRNAs. The details of primers were exhibited in Supplementary Table 1. PCR products of divergent primers were then collected and sequenced to examine the back-splicing sites of circRNAs. We performed RNase R (Geneseed, Guangzhou, China) digestion experiment to detect the stability of circRNAs according to the manufacturer’s instructions. In short, 2 μg RNA was digested in 8 U RNase R for 10 min at 37°C and then repurified it through the RNeasy Mini Kit.
cDNA synthesis and quantitative real-time PCR (Q-PCR)
A total of 1 μg RNA was prepared for reverse transcription with PrimeScript RTreagent Kit (Takara, Tokyo, Japan). QPCR was conducted using a SYBR Green PCR Kit (Takara) following the manufacturer’s instructions. The β-actin was used as reference gene for circRNAs. The qPCR was programed with the CFX 96 q-PCR system (BIO-RAD, Hercules, CA, USA) as follows: 95°C for 5 min; 40 cycles of 95°C for 10 s, 60°C for 34 s. The relative expression of circRNAs was evaluated by 2 −ΔΔ Ct method. The primer sequences information was displayed in Supplementary Table 1.
GO analysis and KEGG pathway analysis of host genes
GO analysis (http://www.geneontology.org) was applied to understand the corresponding host genes of differentially expressed circRNAs. Likewise, following the annotation of the KEGG (http://www.kegg.jp/kegg) [34,35], we disclosed the significant pathways connected with differentially expressed circRNAs.
Statistical Analysis
Results are expressed as the mean ± SEM. The results were analyzed using two-tailed, unpaired or paired Student’s t-test. p<0.05 was considered statistically significant (*=p<0.05/**=p<0.01/***=p<0.001).