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MicroRNA-101 inhibits autophagy to alleviate liver ischemia/reperfusion injury via regulating the mTOR signaling pathway

Liver ischemia/reperfusion injury (LIRI) is a common complication of liver surgery, and affects liver function post-transplantation. However, the precise mechanism underlying LIRI has not yet been completely elucidated. Previous studies have demonstrated the involvement of a number of microRNAs (miRNAs/miRs) in liver pathophysiology. The objective of the present study was to determine the potential function and mechanism of miR-101-mediated regulation of autophagy in LIRI. Compared with the sham-treated group, a significant decrease in miR-101 and mechanistic target of rapamycin (mTOR) expression levels following ischemia/reperfusion (IR) were observed, along with an increased number of autophagosomes (P<0.001). The exogenous overexpression of miR-101 has been demonstrated to inhibit autophagy during the LIRI response and the levels of mTOR and phosphorylated (p)-mTOR expression are correspondingly elevated. However, compared with the miR-NC group, miR-101 silencing was associated with reduced mTOR and p-mTOR levels and increased autophagy, as indicated by the gradual increase in the levels of the microtubule-associated protein 1 light II (LC3II). The peak levels of LC3II were observed 12 h subsequent to reperfusion, which coincided with the lowest levels of miR-101. In addition, inhibition of autophagy by 3-methyladenine significant enhanced the protective effect of miR-101 against LIRI, compared with the IR group (P<0.001). Altogether, miR-101 attenuates LIRI by inhibiting autophagy via activating the mTOR pathway.


Introduction
Liver ischemia/reperfusion injury (LIRI) is a common complication in liver surgery, and substantially influences the surgical outcome and patient prognosis (1)(2)(3). In previous years, the shortage of transplantation donors has increased the prevalence of marginal donors that further increase the risk of LIRI, and subsequently that of primary graft insufficiency or post-transplantation dysfunction (4,5). The mechanisms underlying LIRI pathology are highly complex and include the excessive production of reactive oxygen species, intracellular calcium overload, microvascular endothelial injury, inflammation and autophagy (6). It is essential to elucidate the exact molecular mechanism underlying LIRI, in order to protect the hepatocytes against ischemic injury.
Previous studies have revealed the involvement of microRNAs (miRNAs/miRs) in LIRI-associated autophagy (7)(8)(9). miRNAs are non-coding RNAs ~21-23 nucleotides long, and bind specifically to the 3'-untranslated region (3'-UTR) of the target mRNA, resulting in either mRNA degradation or a protein translation block (10,11). miRNAs may additionally mediate the upregulation of target mRNAs by direct activation and/or indirect derepression to enhance mRNA stability and translational activation (12). miRNAs have been implicated in various cellular and molecular events, including proliferation, differentiation and apoptosis, and the dysregulation of miRNAs is the mechanistic basis of various pathophysiological conditions (13,14). Previous studies have revealed a notable function of miR-101 in modulating autophagy. For example, Frankel et al (15) identified that miR-101 inhibited autophagy in breast cancer cells, whilst Valera et al (16) demonstrated that miR-101 induced multiple system atrophy via autophagy in the nervous system. In addition, Xu et al (17) concluded that miR-101 inhibited autophagy and enhanced cisplatin-induced apoptosis in hepatoma cells. The aim of the present study was to determine the function of miR-101 in mediating autophagy in LIRI, in order to identify a novel therapeutic target for LIRI.

MicroRNA-101 inhibits autophagy to alleviate liver ischemia/reperfusion injury via regulating the mTOR signaling pathway Materials and methods
Animals and cell lines. 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).  (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 random-ized 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.

Reagents and antibodies.
Serological tests. Blood was collected from the mice from their inferior vena cava and centrifuged (4˚C, 15 min, 1,000 x 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).
Hematoxylin and eosin (H&E) staining. 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 x200. IR-induced liver damage was quantified by measuring the Suzuki score as presented in Table I and a previous study (22).
Immunohistochemistry. 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 x200, and ImageJ 1.48v software (National Institutes of Health, Bethesda, Md, USA) was used to analyze the area occupied by the positively stained cells.
Terminal uridine nick-end labeling (TUNEL) assay. 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 x200. The area occupied by the positive cells was analyzed using Image J 1.48v software.
Cell culture and transfection. AML12 cells were plated at a density of 2x10 5 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% cO 2 ) for 12 h to simulate the reperfused (re-oxygenated) state.
Confocal fluorescence microscopy. 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 (3x10 7 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 x1,000. 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.
RT-qPCR. 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'-GTA CAG TAC TGT GAT AAC TGA-3' and reverse, 5'-TGC GTG TCG TGG AGT C-3'), mTOR (forward, 5'-TCG GTG CAA ACC TAC AGA AGC-3' and reverse, 5'-TGC AGG TCG TAT ATG GAC AGA G-3') and GAPDH (forward, 5'-GGA GCG AGA TCC CTC CAA AAT-3' and reverse, 5'-GGC TGT TGT CAT ACT TCT CAT GG-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 = cq target gene -cq reference gene , ΔΔcq = Δcq experimental group -Δcq control group (23).  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. 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.

Results
LIRI alters the expression of miR-101 and mTOR in mouse liver. 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).
LIRI alters serum AST and ALT levels. The levels of serum AST and ALT were significantly increased following reperfusion 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).
LIRI affects apoptosis and autophagy. 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). Histopathological changes in the liver following IRI. 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).

IRI alters proliferation and apoptosis of liver cells. 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. 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 histopathological 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  cantly 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  Yellow dots indicate the autophagosomes with GFP and RFP merging, and the red dots represent the autolysosomes with degraded GFP due to the acidic environment. Scale bars=5 µm. * P<0.05, ** P<0.01 and *** P<0.001 with comparisons shown by lines. (D) Representative immunofluorescence images presenting the expression of p62 in AML12 at x400 magnification. Scale bars=20 µm. miR, microRNA; mTOR, mechanistic target of rapamycin; Nc, negative control; Lc3II, microtubule-associated protein 1 light II; IR, ischemia/reperfusion; GFP, green fluorescence protein; RFP, red fluorescence protein; DAPI, 3,3'-diaminobenzidine. 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).

miR-101 inhibits autophagy and weakens LIRI by activating the mTOR pathway in vitro.
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.
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).

Discussion
Liver transplantation is the only treatment option currently available for end-stage liver disease. Unfortunately, it is associated with various complications, including LIRI, which is a common pathophysiological consequence of liver surgery (26). The mechanism of LIRI is complex, and is closely associated with inflammation, metabolic disorders, oxidative stress and autophagy. In addition, each of these factors may be mutually antagonistic or synergistic (27).
Autophagy is an intracellular self-digestion pathway present in the majority of eukaryotic cells, which helps in organelle recycling and fulfils cellular metabolic requirements under stress conditions (28). The autophagy-related genes (Atgs) induce the detachment of bilayer membrane structures from the rough endoplasmic reticulum, which then encapsulate organelles and other cytoplasmic contents to form autophagosomes. The latter then fuse with lysosomes to form autolysosomes, and the intra-vesicular contents are degraded by the lysosomal enzymes (29). Lc3/Atg8 is a marker of the autophagosome membrane, and the conversion of Lc3I to Lc3II is used as a measure of autophagosome formation (30). One previous study has demonstrated that hepatic autophagy is notably enhanced in LIRI models (31), but the underlying mechanism is not fully understood. Autophagy functions as a double-edged sword in hepatic IR and influences cell survival and apoptosis (32). In moderate IRI, the autophagosomes digest damaged organelles and provide energy to the cells. However, severe reperfusion injury results in excessive autophagy, which may trigger cell death (33). Therefore, targeting the autophagy pathway may effectively protect against IRI (34).
In previous years, studies have focused on the function of miRNAs in hepatic IRI, particularly their involvement in autophagy (35)(36)(37). miRNAs are able to regulate autophagy by inhibiting the expression of target genes, which in turn affect IRI (13). Previous studies have demonstrated that miR-17 upregulated autophagy and aggravated the degree of LIRI by inhibiting Stat3 expression in LIRI (31), while miR-30b reduced autophagy and protected against LIRI by inhibiting Atg12-Atg5 binding (38). Studies have additionally demonstrated that the inhibition of miR-34a enhanced sirtuin 1 expression, which downregulated autophagy and subsequently protected the liver from p65/p53 deacetylation-induced damage (39,40). Therefore, the miRNAs regulating autophagy in LIRI may be potential therapeutic targets. miRNAs typically function by causing mRNA degradation through interacting with the 3'-UTR of the target mRNAs, resulting in mRNA degradation and/or translational repression (12). conversely, the miRNA-mediated upregulation of target mRNAs may be elucidated by direct activation and/or indirect derepression to enhance mRNA stability and translational activation (41). Studies have demonstrated that in miRNA-mediated upregulation, micro-ribonucleoprotein (miRNP) trans-expression promotes the expression of its target mRNA, which is similar to miRNA-mediated downregulation (42)(43)(44). MRNA expression may be activated directly by miRNP and/or alleviated indirectly from miRNA-mediated inhibition by eliminating the inhibitory effect of miRNP (45).
The present study investigated whether miR-101 was able to affect autophagy and serve a function in LIRI through the mTOR pathway. In previous studies, miR-101 was able to inhibit tumor growth by inhibiting autophagy (46)(47)(48). mTOR is a notable serine-threonine protein kinase downstream of phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) (49) and inhibits autophagy during tumor growth and progression (17,47). Li et al (50) revealed that octreotide is able to upregulate the expression of miR-101 and inhibit autophagy by inactivating AMP-activated protein kinase (AMPK) and activating the mTOR pathway, thereby reducing the incidence of intestinal mucositis following anticancer treatment. However, little is known regarding the function of the miR101/mTOR axis in LIRI.
The present study revealed that IRI induced a number of pathological, functional and molecular changes in the liver, including increased serum levels of ALT and AST, the downregulation of miR-101, mTOR mRNA and p62, increased levels of Lc3II and caspase-3, decreased intra-nuclear PcNA and extensive tissue necrosis and apoptosis. PcNA is closely associated with cellular dNA synthesis and therefore a good indicator of cell proliferation status (51,52). caspase-3 is a necessary terminal cleavage enzyme in the intrinsic apoptotic pathway, and an established indicator of apoptosis (53).
Based on the results of the present study, miR-101 was negatively associated with autophagy. Furthermore, the overexpression of miR-101 reduced apoptosis, increased mTOR expression and decreased Lc3II and caspase-3 levels, whilst the inhibition of miR-101 had the reverse effects. In addition, the inhibition of autophagy by 3-MA augmented the protective effects of miR-101 overexpression against LIRI.
To assess the hypothesis that miR-101 regulates the mTOR signaling pathway, the present study investigated the function of miR-101 in the regulation of the mTOR signaling pathway. The overexpression of miR-101 in LIRI-mimicking AML12 cells increased mTOR expression, decreased the number of autophagosomes and increased p62 expression. miR-101 inhibition exhibited the reverse effects on mTOR expression and autophagosome formation. Subsequently, miR-101 and mTOR were co-inhibited in AML12 cells, and it resulted in an increase in autophagy and cell death. These results indicate that miR-101 protects hepatocytes against IR injury by inhibiting autophagy via activation of mTOR signaling pathway.
The present study identified the inhibitory effect of miR-101 on autophagy in LIRI by regulating the expression of mTOR. But, a number of limitations of the present study should be taken into consideration. For example, the underlying mechanisms of the inductory effect of miR-101 on mTOR should be further studied. However, microRNA regulation is multi-directional. Nikoonahad et al (54) revealed that miR-101 inhibits the growth of AML cancer cells by directly upregulating the expression of the pro-apoptotic gene Bcl2 like11 (BIM). different diseases and different conditions may cause miRNAs to exhibit distinct regulatory mechanisms. In addition, as one of the substrates of Akt, the P13K/Akt/mTOR regulatory pathway has been confirmed to serve a necessary function in cell growth and regulation (55). At present, the function of miR-101 in LIRI through the P13K/Akt/mTOR regulatory pathway remains to be further verified. AMPK is a cellular energy receptor, and a number of studies have demonstrated that AMPK is a negative regulator of the mTOR pathway (56,57). Studies have also revealed that miR-101 may inhibit the action of AMPK by directly targeting the 3'-UTR region of AMPK (58,59). Therefore, miR-101 is likely to regulate the mTOR signaling pathway and serve a function in LIRI by affecting the expression of AMPK. Whether miR-101 affects the expression of mTOR by regulating AMPK in LIRI and whether there are other regulatory objectives and mechanisms has yet to be further studied.
In conclusion, the present study revealed that miR-101 attenuates LIRI by activating the mTOR pathway and inhibiting autophagy. Further studies are required to further dissect the association between autophagy and miRNAs in hepatocytes following IR in order to develop novel therapies.