miR-494 up-regulates the PI3K/Akt pathway via targetting PTEN and attenuates hepatic ischemia/reperfusion injury in a rat model

A rat HIRI model was constructed and treated with an intraperitoneal injection of agomir-miR-494 or agomir-NC (negative control) for 7 days after the surgery. The pathophysiological changes in sham-operated rats, HIRI, HIRI + agomir-miR-494, and HIRI + agomir-NC were compared. The effect of miR-494 was also assessed in an H2O2-induced apoptosis model. Hepatic AML12 cells were transfected with mimics NC or miR-494 mimics, followed by 6-h H2O2 treatment. Cell proliferation and apoptosis were detected by CCK8 assay and flow cytometry, respectively. Further, the miR-494 target gene was identified by luciferase reporter assay, and verified both in vitro and in vivo experiments. The activity of AKT pathway was further analyzed in vivo by Western blot. HIRI + agomir-miR-494 rats exhibited significantly higher miR-494 expression, lower serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and glutamate dehydrogenase (GLDH) level, lower hepatic MDA, TOA, and OSI, alleviated hepatic necrosis, reduced hepatocyte apoptosis, and decreased expression of apoptosis-related proteins, when compared with HIRI + agomir-NC rats (P<0.05 or 0.01). After H2O2 treatment, AML-12 cells transfected with miR-494 mimics had significantly higher proliferation and lower apoptosis rate compared with mimics NC group (P<0.01). PTEN was identified as an miR-494 target gene. PTEN expression was significantly down-regulated in AML12 cells transfected with miR-494 mimics, and was up-regulated by treatment of miR-494 inhibitor (P<0.01). Moreover, HIRI + agomir-miR-494 rats exhibited significantly lower PTEN expression, and higher p-AKT, p-mTOR, and p-p70S6K levels compared with HIRI + agomir-NC rats. Therefore, miR-494 protected rats against hepatic ischemia/reperfusion (I/R) injury through down-regulating its downstream target gene PTEN, leading to the activation of PI3K/AKT signaling pathway.


Introduction
Hepatic ischemia/reperfusion (I/R) injury is an inevitable complication occurring during surgical procedures such as partial hepatectomy and liver transplantation [1,2]. During hepatic I/R injury, high levels of reactive oxygen species (ROS) are produced, leading to acute inflammatory responses and hepatocyte apoptosis, which ultimately result in liver dysfunction and even liver failure [3][4][5][6][7][8].
miRNAs are a class of small non-coding RNAs (21-25 nts) that suppress gene expression by binding to the 3 -UTR of the target mRNA [9], and therefore are associated with a variety of biological processes, such as cell differentiation, proliferation and apoptosis, as well as the occurrence and development of diseases [10]. Several studies have suggested the important roles of miRNAs in I/R injury, such as miR-122, miR-124, miR-146a, miR-223, miR-370 [11][12][13][14][15]. Differentially expressed miR-494 has been reported in rats with cerebral I/R injury [16]. miR-494 alleviates the I/R-induced myocardium injury in a mouse model through activating the AKT signaling pathway [17].
Overexpression of miR-494 exerts protective effects against hypoxia/ischemia-induced apoptosis in human hepatic L02 cells via the modulation of PI3K/Akt pathway [18]. However, the potential in vivo role of miR-494 in hepatic I/R injury remains unknown. Therefore, the current study was undertaken to investigate the effect and relevant molecular mechanism of miR-494 in response to I/R-induced hepatic injury in a rat model. We further validated our findings in hepatic AML12 cells treated with H 2 O 2 -induced stress. Our study may provide new insights into the development of novel therapeutic strategies for hepatic I/R injury.

Construction of a rat HI/RI model
Male Sprague-Dawley (SD) rats weighing 180-220 g were purchased from the Slac Laboratory Animals (Shanghai, China) and housed in standard conditions with free access to water and food before this experiment. SD rats were randomly divided into sham, HIRI, HIRI + agomir-NC (negative control), and HIRI + agomir-miR-494 groups (n=6 in each group). Rats in HIRI group were anesthetized by intraperitoneal injection of 3 ml/kg chloral hydrate. Partial (70%) hepatic ischemia was introduced by clamping the artery and portal venous blood supply to the middle and left liver lobes with atraumatic vascular clamps. After 60-min ischemia, the clamp was removed and the liver was reperfused for 6 h. The sham group underwent abdominal surgery without liver I/R. Rats in HIRI + agomir-miR-494 were given an intraperitoneal injection of 20 μl of 500 pmol agomir-miR-494 (GenePharma Biotech, Shanghai, China)/day for 7 days prior to ischemia. Rats in HIRI + agomir-NC were given equal amount of agomir-NC. Rats were immediately killed after the surgery, and blood and left liver samples were collected. The present study was approved by the Research Ethics Committee and performed in strict accordance with the institutional animal care instructions.

Histological examination
Liver samples were fixed in 4% buffered formalin, dehydrated in graded ethanol (70, 80, 90, and 100%), embedded in paraffin, and cut into 4-μm sections. Tissue sections were then dewaxed with xylene, rehydrated in graded ethanol (100, 90, 80, and 70%), and stained with Hematoxylin and Eosin (HE). The pathological changes including inflammatory infiltration and hepatic cell necrosis in five randomly selected visual fields was observed.

TUNEL apoptosis assay
Paraffin sections were dewaxed and hepatic cell apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay using the TUNEL assay kit (Roche, Shanghai, China) according to the manufacturer's protocol. Nuclei were labeled by DAPI staining (Biohao Biotech, Shanghai, China). Sections were then observed under a confocal laser microscope (Olympus, Japan). The percentage of apoptotic cells was calculated by counting TUNEL-positive cells in five randomly selected fields on each slide.

Western blot
Total protein was extracted from harvested liver tissues or cells using lysis buffer (Beyotime, Shanghai, China) and quantitated using BCA Protein Assay Kit (Beyotime) following the manufacturer's instructions. Protein samples were then separated by 10% SDS/PAGE and transferred on to PVDF membranes (GE Healthcare, Amersham, U.K.). Membranes were blocked with 5% skim milk and incubated with appropriate dilutions of primary antibodies overnight at 4 • C as follows: rabbit anti-rat Bax, cleaved caspase-3, cleaved PARP, PTEN, AKT, p-AKT, mTOR, p-mTOR, p70S6K, p-p70S6K, and β-actin. Membranes were then washed with PBST and incubated with goat anti-rabbit HRP-conjugated IgG at room temperature for 1 h. Immunoreactivity was detected using the ECL detection system (GE Healthcare). All antibodies were purchased from Abcam (Cambridge, MA, U.S.A.) or Cell Signaling Technology (Danvers, MA, U.S.A.). β-actin was used as the internal control.

DNA fragmentation assay
The cytoplasmic histone-associated DNA fragmentation in different groups was determined using an ELISA Kit (Roche, Indianapolis, IN, U.S.A.). The experiment was performed in triplicate.

Prediction of miR-494 targets
Both PicTar (http://pictar.mdc-berlin.de) and TargetScan version 6.2 (http://www.target-scanorg/index.html) were used to identify potential downstream targets of miR-494. Genes that were predicted by both databases were considered as potential targets. PTEN, one of the potential targets was selected for further analysis.

Luciferase reporter assay
The PTEN gene was analyzed using the online tool (http://www.targetscan.org) to predict the 3 -UTR sequence for miR-494. The oligonucleotide sequences (3 -UTR of PTEN wild and mutated type) were cloned into the site of a firefly luciferase reporter vector pMIR (Promega, WI, U.S.A.). HEK293T cells at 70% confluence were cotransfected with 500 ng of pMIR-PTEN-wt/pMIR-PTEN-mut and 50 nM of miR-494 mimics/mimics C,G(enePharma Biotech) using a Lipofectamine 3000 transfection kit (Invitrogen). After 24 h, the luciferase activity was measured using a dual luciferase reporter assay system (Promega) following the manufacturer's instructions. The relative luciferase activity (RLA) was calculated as the ratio of firefly luciferase activity (Ff) to Renilla luciferase activity (Rn).

CCK8 detection of cell viability
AML12 cells at 70% confluence were transfected with 50 nM of miR-494 mimics or mimics NC. At 24, 36, and 48 h after transfection, cells were treated with 200 μM H 2 O 2 for 6 h. CCK8 (Beyotime, 10 μl) was added to the cells, and the viability of the cells was measured at 450 nm using a microplate reader.

Annexin V-FITC apoptosis detection
Transfected AML12 cells were treated with 200 μM H 2 O 2 for 6 h. Cells were then collected and resuspended in 500 μl of binding buffer. Annexin V-FITC (5 μl) was added and cell apoptosis was detected by flow cytometry within 30 min.

Detection of apoptosis-related proteins
Transfected AML12 cells were treated with 200 μM H 2 O 2 for 6 h. The expression of cle-PARP, cle-caspase-3, AKT, and p-AKT was determined by Western blot as described above.

Immunohistochemical analysis of apoptosis in liver tissue
Paraffin-embedded sections were dewaxed with xylene, and dehydrated with ethanol. Slices were processed to remove endogenous peroxidase, followed by antigen retrieval and serum blocking. The sections were incubated successively with rabbit anti-rat cleaved caspase-3, biotin-labeled secondary IgG, and horseradish peroxidase labeled streptavidin. Yellow or brown stained cells were positive for cleaved caspase-3.

Statistical analysis
All experiments were performed in triplicate. Data were expressed as mean + − S.D. All statistical analyses were performed using SPSS 16.0 software (IBM SPSS, Chicago, IL, U.S.A.). Differences between two groups were determined by the Student's t test. Differences amongst groups were compared using one-way ANOVA followed by post-hoc Tukey HSD test. P<0.05 was considered as statistically significant.

I/R led to injury of rat liver through induction of oxidative stress and apoptosis
To investigate the potential role of miR-494 in liver I/R injury, we constructed a rat HIRI model. As shown in Figure  1A,B, after the liver I/R surgery, serum level of liver injury indexes including ALT, AST, LDH, and GLDH enzymes was significantly increased compared with sham surgery group (all P<0.01), revealing that I/R induced liver injuries. Moreover, the concentration of oxidative parameters MDA, TOA, and OSI was markedly increased in HIRI group ( Figure 1C). HE staining revealed loss of cell integrity, large areas of hepatocyte necrosis, and inflammatory infiltration in the livers in HIRI group ( Figure 1E). We further evaluated the extent of apoptosis in livers by TUNEL assay, and found that the number of TUNEL-positive cells in HIRI group was notably higher compared with sham surgery group ( Figure 1F). These pathophysiological changes suggested the successful construction of HIRI model in the present study. We then examined the expression levels of miR-494. As shown in Figure 1D, miR-494 in HIRI group was significantly down-regulated at least two fold when compared with sham surgery group (P=0.007).

miR-494 attenuated hepatic I/R injury in rats
In order to analyze the regulatory role of miR-494 in I/R injury, we treated the HIRI rats with an intraperitoneal injection of agomir-miR-494 or agomir-NC. As shown in Figure 2A, although the liver miR-494 concentration in HIRI + agomir-miR-494 group was significantly lower than that in sham surgery group (P=0.046), it was substantially increased compared with HIRI + agomir-NC (P=0.004). Serum ALT, AST, LDH, and GLDH levels in HIRI + agomir-miR-494 group were significantly decreased, as compared with HIRI + agomir-NC group (all P<0.01, Figure  2B,C). Likewise, oxidative parameters of the liver including MDA, TOA, and OSI was significantly reduced compared with agomir-NC treated group (all P<0.01, Figure 2D). Moreover, liver histology from HIRI + agomir-miR-494 rats revealed obvious reduction in I/R-induced hepatocellular necrosis and improvement of cell integrity ( Figure 1E). IHF demonstrated a markedly lower number of TUNEL-positive cells in HIRI + agomir-miR-494 group ( Figure 2E). Immunohistochemical (IHC) results demonstrated a substantially higher expression of cleaved caspase-3 in HIRI and HIRI + agomir-NC groups compared with sham group ( Figure 2F). In contrast, the cleaved caspase-3 expression in HIRI + agomir-miR-494 group was only slightly higher compared with sham group. Consistently, the hepatic levels of cleavage PARP, cleavage Caspase-3, and Bax in HIRI + agomir-miR-494 group was clearly lower compared with agomir-NC group ( Figure 2G). DNA fragment was significantly reduced in agomir-miR-494 treated rats (P=0.001, Figure 2H). All together, these results suggested that miR-494 effectively alleviated the I/R injuries in rat liver via inhibiting the cell apoptosis.

miR-494 decreased H 2 O 2 -induced apoptosis of hepatic AML12 cells by activating AKT
In order to simulate hepatic I/R injury, we further established an in vitro H 2 O 2 -induced apoptosis model. AML-12 cells were treated with H 2 O 2 (0-400 μM) for 6 h, and miR-494 mRNA expression was decreased in a dose-dependent manner when compared with control cells without H 2 O 2 treatment (all P<0.05 or 0.01, Figure 3A). Further, AML-12 cells were transfected with miR-494 mimics or mimics NC, and treated with 200 μM H 2 O 2 for 6 h at all indicated time points. CCK8 assay showed that miR-494 mimics significantly improved cell viability compared with mimics NC group (all P<0.01, Figure 3B). As shown in Figure 3C,D, the apoptosis rate of miR-494 mimics was significantly lower than that in mimics NC group (P=0.002), and the expression of apoptosis-related proteins cle-PARP and cle-Caspase-3 was also significantly reduced (P=0.014 and 0.038, respectively), suggesting that miR-494 decreased H 2 O 2 -induced apoptosis of hepatic AML12 cells. To elucidate the potential mechanism, we further examined the intracellular level of activated AKT, and found that p-AKT expression in miR-494 mimics was obviously higher compared with mimics NC group ( Figure 3E), indicating that the protective effect of miR-494 against H 2 O 2 -induced cell apoptosis was mediated through the activation of AKT.

PTEN is a downstream target gene of miR-494
To identify the downstream target gene of miR-494, bioinformatics analyses (PicTar and TargetScan) were performed. PTEN was identified as one of the candidate targets of miR-494 ( Figure 4A). To validate this prediction, the 3 -UTR  region of PTEN was then cloned into a luciferase system. It was found that miR-494 mimics significantly suppressed the luciferase activity of the 3 -UTR wild-type of PTEN (P=0.006), but not that of the pMIR-PTEN-mut or pMIR group, when compared with the mimics NC group ( Figure 4B). Further, the expression of PTEN mRNA in AML12 cells treated with miR-494 mimics or miR-494 inhibitor was compared by qRT-PCR. As shown in Figure 4C, PTEN   mRNA expression was significantly reduced by miR-494 mimics (P=0.008) and enhanced by miR-494 inhibitor (P=0.000). Consistently, PTEN protein expression was also obviously down-regulated after miR-494 mimics transfection and up-regulated after miR-494 inhibitor transfection ( Figure 4D). Moreover, Western blot revealed that the HIRI rats with lower hepatic miR-494 expression had obviously higher PTEN level in the liver when compared with sham-operated rats, indicating a negative association between miR-494 level and PTEN expression ( Figure 4E). All these findings confirmed PTEN as the downstream target gene of miR-494.

The protective effect of miR-494 in HIRI rats is associated with PI3K/AKT signaling pathway
To further explore the molecular mechanism underlining the protective effect of miR-494 in HIRI rats, we examined the expression of proteins in rat liver, including PTEN, p-AKT, AKT, and the downstream effectors of PI3K/AKT pathway (p-mTOR, mTOR, p-p70S6K, and p70S6K). As shown in Figure 5A,B, the HIRI rats had significantly increased PTEN level in the liver, but decreased p-AKT, p-mTOR, and p-p70S6K compared with sham group (all P<0.01). After agomir-miR-494 treatment, the PTEN level in rat liver was significantly inhibited, and p-AKT, p-mTOR, and p-p70S6K were increased compared with agomir-NC group (P<0.05 or 0.01), indicating that miR-494 protected rat liver against I/R injury through activating the PI3K/AKT signaling pathway.

Discussion
Hepatic I/R injury is one of the major problems during liver surgery, and it greatly affects the surgical outcome(s). Liver I/R injury triggers a rapid release of ROS, leading to hepatocyte injury and apoptosis [19,20]. In the present study, we successfully constructed a rat model of liver I/R injury as indicated by the elevated serum levels of ALT, AST, LDH, and GLDH enzymes, enhanced concentration of hepatic oxidative parameters MDA, TOA, and OSI, extensive hepatocyte necrosis, and increased rate of hepatocyte apoptosis. Differentiated expression of several miRNAs in hepatic I/R injury has been previously reported. For instance, miR-223 was strongly up-regulated in hepatic IRI, whereas miR-122 and miR-146a were markedly down-regulated [11][12][13]. In the present study, we found that miR-494 in HIRI group was significantly down-regulated by at least two fold when compared with sham surgery group. To analyze the regulatory role of miR-494 in hepatic I/R injury, we treated the HIRI rats with an intraperitoneal injection of agomir-miR-494 or agomir-NC, and compared the pathophysiological changes in the liver in the two groups. It was found that agomir-miR-494 significantly decreased serum ALT, AST, LDH, and GLDH levels, liver MDA, TOA, and OSI concentration, hepatocyte apoptosis rate, liver expression of apoptosis-associated proteins (cleaved PARP, cleaved caspase-3, and Bax), hepatocyte necrosis and DNA fragment, when compared with agomir-NC rats. These results suggested that miR-494 effectively alleviated the liver I/R injuries.
The generation of ROS is an essential factor leading to hepatic I/R injury [21,22]. Therefore, cell models with H 2 O 2 -induced oxidative stress have been commonly used to simulate the hepatic I/R injury [14]. In the present study, we further established an in vitro oxidative stress model by H 2 O 2 treatment, and found that the expression of miR-494 in hepatic AML12 cells was dose-dependent decrease. We also showed that transfection of miR-494 mimics significantly improved the proliferation, and reduced apoptosis rate of H 2 O 2 -treated AML12 cells, when compared with mimics NC transfection, indicating that miR-494 exerted protective effects against H 2 O 2 -induced oxidative stress. The PI3K/AKT/mTOR pathway is an important intracellular signaling pathway in the regulation of cell cycle [23]. Studies have shown that the activation of AKT pathway can significantly reduce H 2 O 2 -induced cell apoptosis [24][25][26].
In our study, we found that miR-494 up-regulated the phosphorylation level of AKT in AML12 cells. Moreover, the activity of apoptosis-associated proteins PARP and caspase-3 was decreased after transfection of miR-494 mimics, suggesting that miR-494 reduced the H 2 O 2 -induced apoptosis of AML12 cells via activating the AKT pathway.
To locate the target gene of miR-494, we searched the miRNA target prediction databases PicTar and TargetScan. PTEN was identified as one of the candidate targets of miR-494. Subsequent luciferase reporter assay also confirmed that miR-494 specifically targetted 3 -UTR of PTEN gene. Subsequent in vitro cell experiments showed that PTEN mRNA and protein expression were markedly down-regulated after miR-494 mimics transfection and up-regulated after miR-494 inhibitor transfection, confirming PTEN as a downstream target of miR-494. As a natural inhibitor of the PI3K/AKT pathway, PTEN inhibits the phosphorylation of AKT, and plays an important role in the regulation of cell apoptosis [27,28]. Previous studies have shown that miR-494 can specifically target PTEN, leading to the activation of the PI3K/AKT pathway during various pathophysiologic processes including cell apoptosis, tumor metastasis, and angiogenesis [18,29,30]. The cardioprotective effects of miR-494 against I/R-induced myocardium injury are also dependent on Akt activation [17]. We therefore hypothesized that miR-494 might inhibit the expression of PTEN, which activates the PI3K/AKT pathway and reduces the H/R-induced hepatic apoptosis. To test this hypothesis, we analyzed the activity of PTEN and PI3K/AKT pathway, and found that the HIRI rats had significantly increased PTEN level in the liver, but decreased p-AKT, p-mTOR, and p-p70S6K compared with sham group. After agomir-miR-494 treatment, the PTEN level in rat liver was significantly inhibited, and p-AKT, p-mTOR, and p-p70S6K was increased compared with agomir-NC group (P<0.05 or 0.01). Taken together, miR-494 protected rat liver against I/R injury through activating the PI3K/AKT signaling pathway.
In conclusion, our study showed that miR-494 protected rats against hepatic I/R injury through down-regulating its downstream target gene PTEN, leading to the activation of PI3K/AKT signaling pathway. Our finding may contribute to the development of novel therapies for hepatic H/R injuries.