In total, 20 healthy 5-month-old male Wistar rats (180–220 g) were obtained from the Experimental Animal Center of Harbin Medical University (Harbin, China) and subjected to a 12/12 h light-dark cycle with standard animal room conditions (temperature, 22±1°C; humidity, 55±5%), with food and water available ad libitum. Rats were randomly divided into control and diabetic model (T2DM) groups. Non-diabetic rats were fed a standard diet. Rats with T2DM were gavaged with a high-fat diet (2 ml/day), which contained lard (20%), cholesterol (5%), sucrose (5%), glucose (5%) and salt (6%) emulsified in 20% Tween-80 with 30% propylene glycol in distilled water. After 2 weeks, diabetic rats were intraperitoneally injected with 35 mg/kg/day streptozotocin (STZ; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in 0.1 M citrate buffer (pH 4.3) for 3 days, once a day. Following this, fasting blood glucose (FBG) levels were measured. FBG>16.7 nmol/l was considered to indicate a successfully established diabetic model.

Histopathological alterations and collagen distribution were evaluated by hematoxylin and eosin (H&E) and Masson's trichrome staining. The livers of rats in the control and T2DM groups were quickly excised and fixed in 4% paraformaldehyde for 24 h at 4°C ad subsequently embedded in paraffin. The samples were cut into 5-µm-thick cross-sections and stained with H&E and Masson's trichrome reagent to assess the degree of fibrosis, as previously described (15,25). The degree of fibrosis was quantified with ImagePro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

AML12 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium/Ham's F-12 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), containing 5 µg/ml pre-mixed ITS (insulin, transferrin and sodium selenite; Sigma-Aldrich; Merck KGaA), 40 ng/ml dexamethasone (Sigma-Aldrich; Merck KGaA) and 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences). The AML12 cells were maintained at 37°C with 5% CO₂ and 95% air. When the culture reached 60% confluence, following culturing in serum-free medium for 6 h, the cells were transiently transfected with miRNA-32 mimics (5′-UAUUGCACAUUACUAAGUUGCA-3′), anti-miRNA oligonucleotide (AMO)-32 (5′-UGCAACUUAGUAAUGUGCAAUA-3′) or negative control (5′-UUUGUACUACACAAAAGUACUG-3′) (NC; Guangzhou RiboBio Co., Ltd., Guangzhou, China) at a concentration of 100 nM. X-tremeGENE siRNA Transfection reagent (cat. no. 04476093001; Roche Diagnostics GmbH, Mannheim, Germany) was used as a transfection vehicle.

MTA3-overexpressing pcDNA3.1 (100 nM; IBSBIO, Shanghai, China) and the NC (100 nM; empty pcDNA3.1 plasmid) were transfected into AML12 cells using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions.

Following incubation for 6 h, the culture medium was replaced with fresh medium, as described above, low glucose (LG; 1,000 mg/l) or HG (6,000 mg/l) medium for 24 h at 37°C. Protein and/or RNA was subsequently extracted from cells for further experimentation.

The potential target genes of miR-32 were predicted by TargetScanHuman Release 7.2 (http://www.targetscan.org/vert_72/), miRanda.org (http://miranda.org.uk/) and miRDB (http://www.mirdb.org/).

Luciferase reporters containing the wild-type or mutated 3′-UTR of MTA3 were constructed by using psi-CHECK2 vectors (Promega Corporation, Madison, WI, USA). When the culture reached 90% confluence, the 293T (Cell Bank of Chinese Academy of Sciences, Shanghai, China) cells were seeded in a 24-well plate. When the 293T cells reached 60–70% confluence in a 24-well plate, the 3′-UTR luciferase vectors (100 mg) were co-transfected with miR-32 mimics, AMO-32 or NC using Lipofectamine^® 2000 reagent, according to the manufacturer's protocol. Renilla luciferase reporters (10 ng) were used as an internal control. Following 48 h of transfection, luciferase activity was examined using the Dual-Luciferase Reporter assay system (Promega Corporation), according to the manufacturer's protocol.

Total RNA from rat liver tissues or from AML12 cells was lysed using 1 ml TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The extracted RNA was reverse transcribed into cDNA using a High-Capacity cDNA RT kit (cat. no. 4368814; Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The plates were incubated for 15 min at 16°C, 1 h at 37°C, 5 min at 85°C and finally maintained at 4°C. A SYBR Green PCR Master Mix kit (cat. no. 4309155; Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to quantify the relative levels of E-cad, α-smooth muscle actin (SMA), vimentin, MTA3, Snail and miR-32. GAPDH or U6 were used as an internal control. The cDNA samples were amplified in 96-well plates for 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C, 30 sec at 60°C and 30 sec at 72°C and finally maintained at 4°C. The relative expression of the miRNA and mRNA were determined by the Cq (2^−∆∆Cq) method (26). qPCR was performed on a ABI 7500 FAST Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The sequences of the primers used are presented in Table I.

Protein samples were obtained from liver tissues and AML12 cells using radioimmunoprecipitation assay (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) lysis buffer supplemented with protease inhibitors. Following centrifugation at 12,000 × g for 15 min at 4°C, the supernatant was collected and quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). For the western blot analysis, 100 µg each protein sample was separated by SDS-PAGE (10% gels), transferred to nitrocellulose membranes and blocked for 2 h with 5% non-fat milk at room temperature. Subsequently, the samples were incubated at 4°C overnight with primary antibodies against E-cad (1:1,000; cat. no. ab76055; Abcam, Cambridge, MA, USA), vimentin (1:1,000; cat. no. 7431; Cell Signaling Technology, Inc., Danvers, MA, USA), α-SMA (1:100; cat. no. ab7817; Abcam), MTA3 (1:1,000; cat. no. ab176346; Abcam), Snail (1:500; cat. no. ab82846; Abcam), GAPDH (1:1,000; cat. no. TA-08; ZhongShanJinQiao, Inc., Beijing, China) and collagen-1 (Col-1; 1:1,000, cat. no. ab34710; Abcam) in PBS. Membranes were incubated with a fluorescence-conjugated anti-rabbit immunoglobulin G secondary antibody (1:10,000; cat. no. 926-32211; LI-COR Biosciences, Lincoln, NE, USA) at room temperature for 1 h. The immunoreactivity were detected and quantified using an Odyssey Infrared Imaging System (LI-COR Biosciences) with Odyssey Software (LI-COR Biosciences; version 3.0).

For immunofluorescence staining, AML12 cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature and then treated with 1% bovine serum albumin (cat. no. A-9647; Sigma-Aldrich; Merck KGaA) and 0.4% Triton X-100 (Beijing Solarbio Science & Technology Co., Ltd.) in PBS at room temperature for 2 h. Following blocking with 10% goat serum (cat. no. AR0009; Wuhan Boster Biological Technology Ltd., Wuhan, China), the cells were incubated with primary antibodies against E-cad (1:250; cat. no. ab76055; Abcam) and vimentin (1:100; cat. no. 7431; Cell Signaling Technology, Inc.) in PBS overnight at 4°C. A secondary antibody conjugated with Alexa Fluor 594 (1:500; cat. no. A-11032; Invitrogen; Thermo Fisher Scientific, Inc.) in PBS was added and incubated with the cells for 1 h at 37°C. Nuclei were stained using DAPI (1:500; Beyotime Institute of Biotechnology) for 20 min at room temperature. Immunofluorescence was examined using a confocal laser-scanning microscope (magnification, ×200; Olympus Corporation, Tokyo, Japan).

Total collagen content was detected with a Biocolor Collagen Assay kit (cat. no. S1000; Biocolor Ltd., Carrickfergus, UK) according to the manufacturer's protocol. Briefly, lysates (100 µl) were collected from AML12 cells under HG conditions after 24 h. Sircol dye reagent (1 ml) binding to collagen was added to each sample and the solution was mixed at 4°C for 30 min. Following centrifugation, 1 ml of the alkali reagent was added to each tube to dissolve the sediment. Subsequently, 200 µl of the sample was transferred to a each well of a 96-well plate to measure the absorbance at 540 nm. Total collagen (mg) was calculated using a linear calibration curve generated from standards and normalized to the total protein amount (mg) in each lysate.

A Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used for the detection of cell viability. AML12 cells (60% confluence) were cultured with HG for 24 h. Subsequently, 10% CCK-8 solution was added to the cell culture medium and incubated for 1 h. The optical density was determined at 450 nm on a microplate reader and viability rates were calculated.

Data are presented as the mean ± standard error of the mean. Each experiment was replicated three times independently. Differences between groups were analyzed by one-way analysis of variance followed by Tukey's multiple-comparisons test. Comparisons between two groups were performed using Student's t-test. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

A T2DM rat model was established, accompanied by hyperglycemia. Rats with T2DM exhibited significantly increased blood glucose levels, water intake, blood triglycerides (TG), total cholesterol (TC) and low-density lipoprotein (LDL), as well as decreased body weight and high-density lipoprotein (HDL; Table II). These markers indicated that the T2DM rat model was successfully established in the present study, similar to a previous study (4). It was observed that Col-1 protein and mRNA levels were significantly increased in the T2DM group (Fig. 1A and B). H&E and Masson's trichrome staining of the liver tissues revealed a broadened hepatic portal area, infiltrative inflammatory cells fibers spreading from the portal area and periportal fibrosis in the T2DM group (Fig. 1C). These findings indicated that hyperglycemia induced liver fibrosis, as previously described (6).

To evaluate whether EMT was involved in liver fibrosis, alterations in EMT marker expression were detected. E-cad, an epithelial marker, was markedly decreased. However, mesenchymal markers, including α-SMA and vimentin, increased in expression at the protein and mRNA level in rats with T2DM, compared with the control group (Fig. 1D and E). These results indicated that mesenchymal phenotypes may be involved in liver fibrosis induced by hyperglycemia.

To investigate the effects of HG on EMT in AML12 cells, Col-1, E-cad, α-SMA and vimentin protein (Fig. 2A) and mRNA (Fig. 2B) expression was evaluated. It was observed that Col-1, α-SMA and vimentin expression was significantly upregulated in the HG group, whereas E-cad expression was markedly downregulated, compared with the LG and control groups (Fig. 2A and B). Collagen content was significantly increased in HG-treated AML12 cells (Fig. S1). Normal AML12 cells exhibited a typical epithelial phenotype with a polygonal morphology and tight arrangement. However, exposure to HG for 24 h decreased E-cad (Fig. 2C) and increased vimentin (Fig. 2D) protein expression, compared with the control group, as determined by immunofluorescence. These alterations in E-cad and vimentin expression were in line with the aforementioned results. Hyperglycemic culture exposes cells to a hypertonic microenvironment (27), and cell viability under these conditions was assessed by CCK8 assays. As presented in Fig. S2, HG decreased cell viability compared with the control. These data suggested that HG may have induced EMT in AML12 cells.

As the role of miR-32 and EMT in liver fibrosis remains unclear, miR-32 expression in liver tissue obtained from the T2DM group and in HG-treated AML12 cells was assessed by RT-qPCR. The results demonstrated that miR-32 was significantly upregulated in the liver tissue of the T2DM group (Fig. 3A) and in HG-treated AML12 cells (Fig. 3B), compared with the corresponding control groups. To investigate whether miR-32 was associated with EMT in liver fibrosis, cells were transfected with miR-32 mimics or AMO-32. Transfection efficiency was confirmed by RT-qPCR (Fig. 3C). It was observed that overexpression of miR-32 increased the protein (Fig. 3D) and mRNA (Fig. 3E) expression of Col-1, α-SMA and vimentin, and markedly downregulated E-cad expression (Fig. 3D and E). Additionally, collagen content was significantly increased following overexpression of miR-32 (Fig. S1). These results were supported and confirmed by immunofluorescent staining analysis for E-cad (Fig. 3F) and vimentin (Fig. 3G).

It was investigated whether decreased miR-32 suppressed liver fibrosis by affecting EMT under hyperglycemic conditions. First, AMO-32 transfection efficiency was confirmed by RT-qPCR (Fig. 4A). It was observed that miR-32 inhibition decreased Col-1, α-SMA and vimentin at the protein (Fig. 4B) and mRNA (Fig. 4C) level in HG-treated AML12 cells. However, levels of E-cad were markedly up-regulated under miR-32 inhibition. In addition, a Sircol collagen assay suggested that collagen content was significantly decreased by miR-32 inhibition (Fig. S1). Similar results were observed in immunofluorescence staining results for E-cad (Fig. 4D) and vimentin (Fig. 4E). The results indicated that decreased miR-32 expression may inhibit liver fibrosis by affecting EMT in HG-treated AML12 cells.

To explore the molecular mechanisms by which miR-32 functioned in the progression and metastasis of liver fibrosis, the TargetScan miRNA database was scanned to determine potential gene targets. Various targets were predicted, including MTA3, CXXC-type zinc finger protein 5 and bone morphogenetic protein 5. It was speculated that those involved in EMT may be relevant targets influencing the biological function of miR-32. Of these genes, MTA3 represses the transcription of Snail by binding to the promoter region of Snail. Snail contributes to the repression of E-cad transcription (28). It has been suggested that MTA3 is the major regulator and upstream protein in EMT.

To obtain further direct evidence that MTA3 was a target of miR-32, a binding site for miR-32 was identified on the MTA3 gene (Fig. 5A). In addition, luciferase reporter gene assays demonstrated that miR-32 significant suppressed luciferase activity, when using a vector carrying the wild-type 3′-UTR of MTA3, and a mutation in this binding site attenuated the action of miR-32 (Fig. 5B). Therefore, it was concluded that the inserted MTA3 fragment was a target of miR-32.

Based on the observations of the anti-fibrotic functions of decreased miR-32 expression and the bioinformatics analysis, the effects of miR-32 on MTA3 expression was investigated. MTA3 expression in the liver tissue and AML12 cells was assessed. Protein (Fig. 5C) and mRNA (Fig. 5D) expression of MTA3 was significantly decreased in liver tissues of diabetic rats and in HG-treated AML12 cells, suggesting that MTA3 may be involved in hyperglycemia-induced liver fibrosis. Western blotting and RT-qPCR were performed to determine the effects of miR-32 on endogenous MTA3 protein and mRNA expression in AML12 cells. It was observed that the overexpression of miR-32 in AML12 cells markedly downregulated MTA3 protein and mRNA (Fig. 5E).

To confirm whether miR-32 regulated liver fibrosis through MTA3 changes in MTA3 protein and mRNA levels were assessed following miR-32 knockdown. In addition, EMT markers in MTA3 overexpressing HG-treated AML12 cells were evaluated. It was observed that miR-32 knockdown by AMO-32 increased MTA3 protein and mRNA levels compared with control and miR-NC cells (Fig. 5F). In addition, E-cad mRNA and protein levels were markedly increased by MTA3 overexpression. However, MTA3 overexpression decreased α-SMA and vimentin protein and mRNA levels at increased MTA3 levels in HG-treated AML12 cells (Fig. S3A and B). The transfection efficiency of MTA3-overexpressing plasmid was confirmed by western blotting (Fig. S3C). In conclusion, these findings indicated that miR-32 knockdown may ameliorate liver fibrosis progression and metastasis via the regulation of MTA3 expression.

Previous studies have suggested that Snail, an EMT-associated zinc finger transcription factor, is a transcriptional target of MTA3 (29,30). Therefore, it was hypothesized that Snail may be involved in the inhibition of liver fibrosis induced by decreased miR-32 expression. In the present study, Snail mRNA and protein expression was significantly increased in liver tissue of rats with T2DM and in HG-treated AML12 cells, compared with their respective controls (Fig. 6A and B). To further explore whether miR-32 indirectly regulated the participation of Snail in EMT, gain- and loss-of-function studies were performed. Treating the cells with a miR-32 mimic reduced Snail expression, as demonstrated by RT-qPCR. Furthermore, miR-32 overexpression decreased MTA3 protein expression in AML12 cells, and miR-32 inhibition by AMO-32 reduced Snail protein and mRNA in HG-treated AML12 cells (Fig. 6C and D).