A total of 60 male C57BL/6 mice (7–8 weeks old, weighing 20–25 g) were obtained from the Experimental Animal Center of Academy of Military Medical Sciences (Beijing, China). All animals were maintained in an air-conditioned animal room at 25°C with free access to water and food, and exposed to a 12-h light/dark cycle. All animal experiments conformed to the National Institute of Health guidelines (18,19), and the animals were treated humanely. The study passed the ethical review of the Tianjin First Center Hospital (Tianjin, China) for the use of experimental animals, and the protocols were ethically approved by the ethics committee of Tianjin First Central Hospital. The non-tumorigenic mouse hepatocyte acute myeloid leukemia (AML)12 cell line was purchased from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China).

Fetal bovine serum, 0.05% trypsin-ethylenediaminetetraacetic acid and Dulbecco's modified Eagle's medium (DMEM)/F12 medium were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The miR-101 mimetics/inhibitor, miR-101 agomir/antagomir, miRNA negative control (miR-NC), and RiboFECTTM CP Reagent were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Trizol and SYBR Green reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Master Mix were purchased from Invitrogen (Thermo Fisher Scientific, Inc.) and Beijing Transgen Biotech Co., Ltd. (Beijing, China), respectively. The In Situ Cell Death Detection kit was purchased from Roche Diagnostics GmbH (Mannheim, Germany). An immunohistochemistry kit (cat. no. PV-9001) and DAB chromogenic kit (cat. no. ZLI-9018) were purchased from OriGene Technologies, Inc. (Beijing, China). The autophagy double-labeled adenovirus [m red fluorescence protein (RFP)-green fluorescence protein (GFP)-LC3] was acquired from Hanbio Biotechnology Co., Ltd. (Shanghai, China), and 3-methyladenine (3-MA) from Selleck Chemicals (Houston, TX, USA). Rapamycin (Rapa) and methylthiazole tetrazolium kit (MTT) were acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies against mechanistic target of rapamycin (mTOR; cat. no. 2972), phosphorylated (p-)mTOR (cat. no. 2971), caspase-3 (cat. no. 9662), sequestosome 1/p62 (cat. no. 16177), microtubule-associated protein 1 light II (LC3II; cat. no. 3868), proliferating cell nuclear antigen (PCNA; cat. no. 13110) and GAPDH (cat. no. 5174), and the horseradish peroxidase (HRP)-conjugated anti-rabbit (cat. no. 7074) and anti-mouse (cat. no. 7076) secondary antibodies were all purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA).

This experiment established a segmental (70%) LIRI model according to a previous study (20), with the arterial and portal venous blood supply to the left and middle lobes interrupted using an atraumatic clip. Following 90 min of local ischemia, the clip was removed. Animals were sacrificed by dislocation of spine and harvested after 2, 6, 12 or 24 h reperfusion. Sham-operated mice underwent the same procedure, but without vascular occlusion as previous described (20). The mice were randomized into the following 10 groups (n=6/group): A control/sham operated group, 4 untreated LIRI groups with different reperfusion times (2, 6, 12 and 24 h) and 5 LIRI groups that were administered an intravenous injection 24 h prior to ischemia and harvested subsequent to 12 h reperfusion, with injections consisting of the following: i) 10 nM miR-101 agomir, ii) 10 nM miR-101 antagomir, iii) 10 nM miR-NC, iv) 5 mg/kg 3-MA and v) miR-101 agomir plus 3-MA. The 3-MA was intraperitoneally administered 1 h prior to ischemia.

Blood was collected from the mice from their inferior vena cava and centrifuged (4°C, 15 min, 1,000 × g) to collect the serum. The levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and according to the manufacturer's protocol. Enzyme activity was expressed as international units per liter (U/I).

The liver tissues were fixed in 4% formalin for 48 h at 4°C, embedded in paraffin blocks and processed into 4-µm-thick sections. The slides were then dehydrated using an ethanol gradient (100% for 10 min, 95% at 10 min and 80% for 10 min) and de-paraffinized using xylene. H&E staining was performed according to a standard procedure (21), and the histopathological changes were observed under a light microscope at a magnification of ×200. IR-induced liver damage was quantified by measuring the Suzuki score as presented in Table I and a previous study (22).

Paraffin sections of liver tissue were cut into 4-µm-thick sections and dehydrated, cleared using xylene and heated to 95°C using 0.01 mol/l citrate buffer solution (pH 6.0) in a water bath for antigen retrieval. Subsequent to blocking with 5% goat serum (Solarbio Science & Technology Co., Ltd., Beijing, China) for 1 h at room temperature, the sections were incubated with rabbit anti-mouse PCNA and caspase-3 polyclonal antibodies (both dilution, 1:1,000; Cell Signaling Technology, Inc.) overnight at 4°C. Then sections were incubated with enzyme-labeled goat anti-rabbit immunoglobulin G (IgG) polymer from the immunohistochemistry kit for 1 h at room temperature. In total, 0.5 ml DAB staining solution A, 0.5 ml DAB staining solution B and 1 ml DAB staining working solution were prepared and gently mixed. The sections were incubated with the mixture at 20–25°C for 5 min, and the sections were counterstained with 10% hematoxylin for 30 sec at room temperature. The stained slides were washed thoroughly in running tap water, dehydrated and mounted with cover slips. Hepatocytes positively stained for PCNA and caspase-3 were examined under a light microscope at a magnification of ×200, and ImageJ 1.48v software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the area occupied by the positively stained cells.

The In Situ Cell Death Detection kit (with TMR red as the fluorescence marker) was used to detect TUNEL positive apoptotic cells according to the manufacturer's protocol. Apoptosis was observed under fluorescence microscope at a magnification of ×200. The area occupied by the positive cells was analyzed using Image J 1.48v software.

AML12 cells were plated at a density of 2×10⁵ cells/ml in 6-well plates and divided into 4 treatment groups, as followings: i) A control untreated group; ii) an ischemia/reperfusion (IR) group subjected to hypoxia for 1 h to simulate ischemia and then cultured in DMEM/F12 for 12 h to simulate reperfusion; iii) an miR-101 mimetics group transfected with 50 nM miR-101 mimetics or miR-NC using RiboFECTTM for 48 h at 37°C, and subjected to hypoxia/reperfusion 48 h later; and iv) miR-101 inhibitor group transfected with 50 nM miR-101 inhibitor or miR-NC, and subjected to hypoxia/reperfusion 48 h later. To induce hypoxia, the culture medium was removed 48 h after transfection and replaced with 2 ml Hank's solution (Thermo Fisher Scientific, Inc.), and the cells were placed in a low oxygen incubator. After 1.5 h, Hank's solution was removed and replaced with 2 ml DMEM/F12 medium, and the cells were cultured under normal oxygen tension (with 5% CO₂) for 12 h to simulate the reperfused (re-oxygenated) state.

AML12 cells were plated in 24-well plates, and cultured until they reached 60–70% confluence. The cells were transduced with the GFP-RFP-LC3 adenovirus (3×10⁷ PFU/well) for 48 h at 37°C according to the manufacturer's protocol to induce autophagy and the resulting autophagosomes were observed under a confocal microscope at a magnification of ×1,000. The number of autophagosomes in each cell was counted, and the mean number of autophagosomes of all cells was calculated to determine the autophagic degree of each treatment group.

Total RNA was extracted from tissues or cells using TRIzol total RNA isolation reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequently, the PrimeScript RT reagent kit (Beijing Transgen Biotech Co., Ltd.) was used for RT of the RNA into cDNA, according to the manufacturer's protocol. SYBR-Green RT-qPCR Master Mix was used as the flurophore. RT-qPCR was performed using specific primers for miR-101 (forward, 5′-GTACAGTACTGTGATAACTGA-3′ and reverse, 5′-TGCGTGTCGTGGAGTC-3′), mTOR (forward, 5′-TCGGTGCAAACCTACAGAAGC-3′ and reverse, 5′-TGCAGGTCGTATATGGACAGAG-3′) and GAPDH (forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′) used as the internal control. The thermocycling conditions were as follows: Pre-denaturation at 94°C for 30 sec, followed by 45 cycles of denaturation at 94°C for 5 sec, annealing at 60°C for 15 sec and extension at 72°C for 10 sec. Each sample was tested in quadruplicates. The relative expression of each gene was quantified using the comparative quantification cycle method as follows: Copy number of target gene = 2^−ΔΔCq, ΔCq = C_{qtarget gene}−Cq_{reference gene}, ΔΔCq = ΔCq_{experimental group}−ΔCq_{control group} (23).

AML12 cells were transfected with miR-101 mimetics or miR-101 inhibitors using RiboFECTTM for 48 h at 37°C, according to the reagent manufacturer's protocol. Following differentiation and treatments, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. Cells were then incubated for 60 min at room temperature with blocking solution (5% goat serum) followed by overnight incubation at 4°C with anti-p62 antibodies (dilution, 1:800) Following washing with PBS, cells were incubated with fluorescence-labeled secondary antibodies (Alexa Fluor^® 594-conjugated goat polyclonal anti-rabbit; 1:500; cat. no. 8889; Cell Signaling Technology, Inc.) for 1 h at room temperature in the dark. In addition, DAPI (1:1,000; cat. no. D9564; Sigma-Aldrich; Merck KGaA) was used to non-specifically stain the nuclei and samples were incubated with 50 µl DAPI for 10 min at room temperature. Immunostaining was visualized under a fluorescence microscope at a magnification of ×400.

AML12 cells were seeded into 96-well plates (5×10⁴ cells/well) and after 24 h culturing, were treated with Rapa or 3-MA for 2 h, or miR-101 inhibitor for 48 h at 37°C prior to reperfusion. Fresh medium was then added to each well along with 20 µl MTT solution (5 mg/ml), and the cells were incubated for another 4 h at 37°C. The medium was then removed, and 200 µl dimethylsulfoxide was added per well to stop the reaction. The optical density of each well was determined at 490 nm.

Radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) were used to extract the total protein from AML12 cells and liver tissues. The protein concentration was determined by Bicinchoninic Acid Protein Assay kit (Solarbio Science & Technology Co., Ltd.). Equal samples of protein (30 µg) were separated by SDS-PAGE on 12% gels and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk for 1 h at room temperature. The membrane was then incubated with mTOR, p-mTOR, GAPDH, LC3 II, caspase-3 and p62 (all 1:1,000) primary antibodies overnight at 4°C. The membranes were then washed with TBS-Tween-20 at room temperature (5 min/wash). Subsequently, the membrane was treated with HRP-conjugated goat anti-rabbit and anti-mouse IgG secondary antibodies (both 1:2,500), agitated and incubated at room temperature for 1 h. The protein bands were visualized by using a G:BOX imaging system (Gene Company, Ltd., Hong Kong, China). The protein bands were measured with ImageJ 1.48v software and normalized to the corresponding GAPDH bands. The relative density of each target protein normalized to the control was used to represent the changes in expression of target proteins.

Statistical analysis was performed using SPSS 22.0 software (IBM Corp., Armonk, NY, USA). Normally distributed data were expressed as the mean ± standard deviation (x±s). A Student's t test was used for comparing two groups, a one-way analysis of variance for comparing multiple groups and the Least-Significant-Difference method was used as a post-hoc test for multiple comparisons between groups. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of miR-101 and mTOR mRNA initially significantly increased following reperfusion compared with their respective sham groups (P<0.001), but significantly decreased in the liver of the IR mouse model following reperfusion at 6 and 12 h compared with the respective sham groups (P<0.001; Fig. 1A).

The levels of serum AST and ALT were significantly increased following reper-fusion in a time-dependent manner compared with the sham groups (P<0.001). Compared with the sham-treated group, the IR mice exhibited a gradual increase in the serum AST and ALT levels that peaked at 12 h (P<0.001; Fig. 1B).

As presented in Fig. 1C, LC3II expression levels significantly increased following 6 and 12 h reperfusion and peaked at 12 h compared with the sham group (P<0.01) and the pro-apoptotic caspase-3 was also significantly upregulated following reperfusion with maximum expression at 12 h post-reperfusion compared with the sham group (P<0.05). Although the expression levels of mTOR and p62 increased after 2 h reperfusion, the expression levels of mTOR, p-mTOR and p62 significantly decreased steadily in a time-dependent manner following reperfusion at 6, 12 and 24 h (P<0.05).

The liver tissues of the IR mice demonstrated edema, ballooning, steatosis, flaky necrosis, neutrophil infiltration and congestion, in addition to the disappearance of the hepatic sinusoidal structure in certain areas. These lesions were substantially aggravated with time, with the severest injuries observed at 12 h after reperfusion, but were relieved at 24 h post-reperfusion. In addition, IR-induced liver damage was quantified by measuring the Suzuki score, which gradually significantly increased following reperfusion compared with the sham group (P<0.01; Fig. 2A).

Compared with the sham-treated group (Fig. 2B), the intra-nuclear expression of the proliferative marker PCNA significantly decreased following reperfusion at the 6 and 12 h mark, despite initially increasing at the 2 h mark and again increasing at the 24 h mark (P<0.01). The cytoplasmic expression of caspase-3 gradually significantly increased in the liver cells of IR mice with time compared with the sham group (P<0.001) and a peak change was observed 12 h after reperfusion. Additionally, as presented in Fig. 2C, the number of TUNEL-positive apoptotic cells were also significantly higher in the IR groups compared with the sham-treated group (P<0.05). The apoptotic changes were time dependent, with peak alterations observed 12 h after reperfusion.

As presented in Fig. 3A, the expression levels of mTOR in the miR-101 agomir group were significantly increased compared with the mTOR miR-NC group (P<0.001), while the expression levels of mTOR in the mir-101 antagomir group were significantly decreased compared with the mTOR miR-NC group (P<0.01; Fig. 3A). The miR-101 antagomir significantly aggravated the histopatho-logical changes in the liver and the corresponding Suzuki scores induced by IR treatment, while miR-101 agomir significantly alleviated these changes compared with the miR-101 miR-NC group (P<0.001; Fig. 3B). The overexpression of miR-101 significantly reduced the expression of LC3II and caspase-3 compared with the miR-101 miR-NC group (P<0.001) and significantly increased that of mTOR compared with the miR-101 miR-NC group (P<0.01; Fig. 3C). IR-induced apoptosis was significantly increased by miR-101 antagomir compared with the miR-NC group (P<0.05) and alleviated by miR-101 agomir compared with the miR-NC group (P<0.01; Fig. 3D).

Treatment of the IR mice with the autophagy inhibitor 3-MA in addition to miR-101 transfection significantly reduced the IR-induced histopathological changes and Suzuki scores compared with the IR group (P<0.001; Fig. 4A) and significantly increased the nuclear expression of PCNA compared with the untreated IR group (P<0.001; Fig. 4B). In addition, IR-induced apoptosis was significantly reduced in the mice treated with miR-101+3-MA compared with the untreated IR group (P<0.001; Fig. 4C) and validated by the significantly lower expression levels of caspase-3 and LC3II and the upregulation in p62 levels compared with the IR group (P<0.001; Fig. 4D). Serum AST and ALT levels were also significantly lower in the miR-101+3-MA group compared with the IR group (P<0.001; Fig. 4E).

As presented in Fig. 5, the expression levels of mTOR in the miR-101 mimetics group were significantly increased compared with the mTOR NC group (P<0.001), while the expression level of mTOR in the miR-101 inhibitor group was significantly decreased compared with the mTOR NC group (P<0.01; Fig. 5A). In addition, the overexpression of miR-101 significantly reduced the expression of LC3II and caspase-3 compared with the NC group (P<0.05; Fig. 5B). The Ad-GFP-RFP-LC3 system was used to determine the potential function of miR-101 in modulating autophagy subsequent to simulated-IR in AML12 cells. The presence of co-localized GFP-LC3 or RFP-LC3 granules indicate the recruitment of the LC3 protein to autophagosomes, which are formed when autophagy is triggered (24). When autophagosomes fuse with lysosomes and form autolysosomes, GFP but not RFP degrades in the acidic environment, resulting in solely red granules (25). As presented in Fig. 5C, the number of autophagosomes in the miR-101 mimetics group was significantly lower compared with that in the IR group (P<0.001), indicating that miR-101 inhibits autophagy. Furthermore, the number of autophagosomes significantly increased upon miR-101 inhibition compared with that in the IR group (P<0.05). In addition, p62 was upregulated in the miR-101 mimetics group and was downregulated in the miR-101 inhibitor group compared with the NC group (Fig. 5D). Altogether, the overexpression of miR-101 inhibited the formation of autophagosomes and autolysosomes, and thus attenuated autophagy.

Reperfusion was established following the pre-treatment of AML12 cells with miR-101 inhibitor and mTOR inhibitor rapamycin. The expression of LC3II and caspase-3 were significantly increased compared with the IR group (P<0.001; Fig. 6A), and the percentage of viable cells was significantly decreased following co-suppression compared with the IR group (P<0.001; Fig. 6B).