Materials and reagents
Antibodies used in this research include rabbit anti-keap1 (cat. #ab227828, Abcam, Cambridge, UK); rabbit anti-Nrf2 (cat. #ab62352, Abcam, Cambridge, UK); rabbit anti-heme oxygenase 1 (cat. #ab1889491, Abcam, Cambridge, UK); rabbit anti-NQO1 (cat. #11451-1-AP, Proteintech Group, Inc, Rosemont, USA); anti-Bax (cat. #50599-2-lg, Proteintech Group, Inc, Rosemont, USA), anti-Bcl-2 (cat. #3498, CST, Danvers, MA, USA) and anti-cleaved caspase-3 (cat. #9661, CST, Danvers, MA, USA); rabbit anti-GAPDH (cat. #AB0037, Abways Technology, Shanghai, China); rabbit anti-Lamin B (cat. #WL01775, Wanleibio, Shanghai, China). Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Beyotime Biotechnology (cat. #S0033S, Shanghai, China).
Serum collection
Whole blood from 27 liver transplantation patients was collected preoperatively, 4 h after reperfusion, and on days 1, 2, and 3 after surgery. Blood was collected allowed to stand and clot at room temperature (RT). Then the tube was centrifuged at 3000 g for 15 min at 4°C, and stored at -80°C. All procedures were approved and were performed with the patient’s informed consent, and this research was approved by the human ethics committee of the First Affiliated Hospital of Chongqing Medical University.
Construction of the hepatic I/R injury model
C57BL/6J mice (male, 18-20 g, 6-8 months old) were purchased from Chongqing Medical University Experimental Animal Center (Chongqing, China). Animals were maintained in a specific pathogen-free (SPF) setting with 12 h light/12 h dark conditions. All animal experiments were approved by the institutional animal care and use committee.
A warm partial (70%) liver I/R injury model was established as previously described [18]. After anesthesia with the sodium pentobarbital (intraperitoneal injection, 50 mg/kg), we opened the abdomen and clamped the left liver and middle liver artery. The clamp was removed after 60 min of ischemia and then reperfusion was performed (reperfusion time: 0 h, 1 h, 3 h, 6 h, 9 h) (n=5 in each time point). The control group was the sham operation group (n=5). Mice were euthanized with an overdose of sodium pentobarbital (100 mg/kg intravenous). Subsequently, the liver tissues and 1 ml venous blood were harvested for subsequent experiments.
Overexpression of miR-141-3p in vivo
Mouse pre-miRNA-141-3p lentivirus gene transfer vectors were constructed by Hanbio (Shanghai, China). The pre-miRNA-141-3p lentivirus was prepared at 1×108 transfection units/ml according to the instructions. Mice were divided into 4 groups: sham, I1R6, I1R6+vector control and I1R6+miR-141-3p. The pre-miRNA-141-3p lentivirus and NC lentivirus were transfected with approximately 2×107 transfection units in vivo by tail vein injection into mice.
Cell culture
The human liver LO2 cell line was purchased from Zhong Qiao Xin
Zhou Biotechnology Co., Ltd (Shanghai, China). Cells were incubated in RPMI 1640 basic medium (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (Biological Industries, Israel). For the H/R model, cellular hypoxia was induced by incubation in serum-free medium and culturing cells in a tri-gas incubator (Thermo, MA, USA) with 94% N2, 1% O2, and 5% CO2. Cells were exposed to hypoxia for 2 h, 4 h, 8 h, 12 h and 24 h followed by reoxygenation for 6 h.
Cell transfection
Sequences of miRNA-141-3p mimic and inhibitor were as follows: miRNA-141-3p mimic sense 5′-UAACACUGUCUGGUAAAGAUGG-3′, miRNA-141-3p inhibitor sense 5′-CAGUACUUUUGUGUAGUACAA -3′. MiRNA-141-3p mimic and miRNA-141-3p inhibitor were diluted in serum-free medium, as well as the Lipofectamine 2000 (Invitrogen, Carlsbad, USA) transfection reagent, and then cells were transferred to cell culture medium after mixing. Six hours later, the medium was replaced. Total RNA was extracted 24-48 h after transfection.
Hematoxylin and eosin (HE) staining
After the left lobe of the liver was obtained, part of the liver tissue was fixed in 10% buffered formalin. After deparaffinization and rehydration, hematoxylin/eosin staining and mounting were performed and sections were observed at 200× magnification.
Reverse transcription‑quantitative PCR (RT‑qPCR) analysis
Total RNA extraction from serum was performed using a miRcute serum/plasma miRNA isolation kit (Tiangen, Beijing, China). Total RNA was extracted from liver tissues and cells with TRIzol reagent (Takara Bio Inc, Japan). The primers U6, Keap1 and GAPDH primers were purchased from Takara. The primer sequences of miR-141-3p (RiboBio Co., Guangzhou, China) are proprietary information from the company. U6 primer: forward 5’-AGAGAAGATTAGCATGGCCCCTG-3’ and reverse 5’-ATCCAGTGCAGGGTCCGAGG-3’. Keap1 primer: forward 5’- CCCAATGCTGACACGAAGG-3’ and reverse 5’- GCTGAATTAAGGCGGTTTGTC-3’. GAPDH primer: forward 5’-CACTCCTCCACCTTTGACGC-3’ and reverse 5’-CTGTTGCTGTAGCCAAATTCGT-3’. Relative expression level was calculated using the 2-∆∆Ct method.
Enzyme-linked immunosorbent assay (ELISA)
Serum samples were collected from patients as described above. Interleukin-6 (IL-6) (4A Biotech, China) and interleukin-1β (IL-1β) (NeoBioscience, China) concentrations in the serum were determined by ELISA kits according to the instructions.
Western blot analysis
Total protein and nuclear protein were extracted primarily using RIPA and nuclear protein extraction kit (Beyotime, Shanghai, China). Protein concentration was measured by BCA (Beyotime, Shanghai, China). After separation in gels, blocking was performed for 15 min using quick western blocking solution. After overnight incubation with primary antibodies (1:1,000 with diluent), proteins were combined with the primary antibody. Then, we incubated the proteins with secondary antibody (1:10,000 diluent) for 1.5 h. We visualized the proteins using Fusion FX7.
Luciferase reporter assay
The 3’-UTR of Keap1 was amplified and cloned into the pGL3 vector (Promega, WI, USA), creating wild-type (WT) pGL3-Keap1 3’-UTR. Point mutations in the potential miR-141-3p binding site were performed to create mutated pGL3-Keap1 3’-UTR. For the luciferase assay, LO2 cells were transfected with pGL3-Keap1 3’-UTR-wt/mutant and miR-141-3p mimic/NC. Luciferase activities were measured using the Dual Luciferase kit (Promega, WI, USA).
Measurement of intracellular ROS accumulation
DCFH-DA fluorescence dye was used to examine intracellular ROS accumulation. Briefly, LO2 cells were seeded in plates and treated with the miRNA-141-3p mimic and inhibitor. After incubation, DCFH-DA was added to serum-free culture medium, which was added to the cells and then incubated for 15 min in the dark at 37°C. For flow cytometry analysis, dichlorofluorescein (DCF) fluorescence intensity was measured using a BD FACSAria II flow cytometer (USA). For confocal laser scanning microscopy (CLSM), after DCFH-DA staining, cells were incubated with Hoechst for 15 min at 37°C, and ROS levels were observed under CLSM (ZESS, Germany) at 488 nm by comparing the fluorescence intensity (green signal).
Serum levels of aminotransferase
Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are indexes of liver injury. ALT and AST assay kits (Nanjing Jiancheng, China) and microplate readers (Biotek, USA) were used to measure mouse serum ALT and AST levels.
Statistical analysis
Data are expressed as the mean ± standard deviation (SD). Differences between groups were analyzed using one-way analysis of variance (ANOVA). Correlation analysis was performed using Spearman’s rank correlation method. SPSS 18.0 software (SPSS, Chicago, IL, USA) was used to perform statistical analyses, and p < 0.05 was considered statistically significant.