A total of 60 male Sprague Dawley rats (aged 6–8 weeks; weighing, 180–220 g) were purchased from Laboratory Animal Services Centre of Hunan Slack Jingda Experimental Animal Co., Ltd. (Changsha, China). Rats were housed at 18–22°C with 12-h light/dark cycles and access to standard food and water ad libitum. All procedures were approved by the Animal Ethics Committee of Second Affiliated Hospital of Nanchang University (Nanchang, China). Rats were fed with a high glucose, high fat diet (60% standard chow, 10% lard, 10% egg yolk powder and 20% sucrose). Following 8 weeks, rats were divided into two groups: The diabetic group (n=50) and the control group (n=10). Rats in the diabetes group were injected with STZ (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 40 mg/kg. At 72 h following STZ treatment, rats were considered diabetic providing their fasting blood glucose levels were >16.7 mM (21). The control group received 2 ml of 0.1 M sodium citrate buffer.

Anti-miR-204-5p [adenovirus AAV-U6-rno-miR-204-5p-CAG-enhanced green fluorescent protein (EGFP); cat. no. MICA2014002038], miR-204-5p mimic (adenovirus AAV-U6-mimic-rno-miR-204-5p-CAG-EGFP, 5′-UUCCCUUUGUCAUCCUAUGCCU-3′) and negative control miRNA (neg-miR, adenovirus AAV-U6-mimic-rno-negative-miR-CAG-EGFP, 5′-UUCUCCGAACGUGUCACGUTT-3′) were obtained from Shanghai GeneChem Co., Ltd. (Shanghai, China). Diabetic rats were randomly divided into three groups: Neg-miR, anti-miR-204-5p and mimic groups. Diabetic rats in the anti-miR-204-5p group (n=15) were injected with anti-miR-204-5p adenovirus (4.1×10¹² viral genome/ml) via the caudal vein. Diabetic rats in the mimic group (n=15) received miR-204-5p mimic adenovirus (4.1×10¹²viral genome/ml). The neg-miR group (n=15) were injected with the same quantity of neg-miR adenovirus. At 8 weeks, retinal tissues were extracted for further investigation as detailed below.

Five diabetic rats were anesthetized by intraperitoneal injection of pentobarbital sodium (30 mg/kg). Under anesthesia, the eyeball of diabetic rats was isolated from diabetic rats and following rats were sacrificed using a lethal dose of pentobarbital (100 mg/kg body weight). The retina was separated from the eyeball and washed with Hanks balanced salt solution (H1020; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and minced into small pieces (1×1 mm) with surgical scissors. Following filtration through a 30-µm nylon sieve, the retina was digested with 2.5% trypsin for 30 min at 37°C. Following centrifugation (1,500 × g for 5 min) at room temperature, rRECs were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 20% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA) at 37°C with 5% CO₂. Medium was replaced every three days. rRECs were identified for endothelial homogeneity by testing for immunoreactivity to factor VII antigen. rRECs were fixed with 4% paraformaldehyde, blocked with 2% BSA for 1 h at room temperature and incubated with mouse anti-factor VII antibody (cat. no. ab97614; Abcam, Cambridge, UK) at 4°C overnight. The cells were washed and incubated with Alexa Fluor^® 647-conjugated donkey anti-rabbit immunoglobulin G secondary antibodies (cat. no. ab150075; Abcam) at 37°C for 2 h. Then nuclear was stained with DAPI (Sigma-Aldrich; Merck KGaA) for 10 min at room temperature. Signals were detected with a fluorescent inverted microscope at a magnification of ×200. The third passage was prepared for subsequent experiments.

rRECs were seeded in 6-well plates at 1×10⁶ cells/well and cultured in DMEM supplemented with 10% FBS. At 70–80% density, cells were transfected with 100 nM miR-204-5p mimic (the mimic group), miR-204-5p inhibitor (the anti-miR-204-5p group) or neg-miR (the neg-miR group) with Lipofectamine 3000 (Roche Diagnostics, Basel, Switzerland). The transfection efficiency was determined by RT-qPCR. At 48 h, cells were collected for follow-up experiments.

rRECs at a density of 1×10⁵ cells/well were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 for 2 h at room temperature. Following washing with 0.1 M phosphate buffer, samples were stained with 1% osmium tetroxide for 1 h at room temperature. Following dehydration with an ethanol gradient, samples were embedded in LX-112 resin and heating at 70°C overnight. Sections were sliced to ultrathin sections (70 nm) and imaged with an FEI Tecnai Spirit transmission electron microscope (FEI; Thermo Fisher Scientific, Inc.) at magnifications of ×1,700 and ×5,000

Total RNA was isolated from retinal tissues and rRECs using TRIzol reagent (cat. no. CW0580S; Beijing CWBio, Beijing, China) according to the manufacturer's protocol. RNA quality was evaluated by agarose electrophoresis and the quantity was determined via spectrophotometry at 260 and 280 nm. A total of 500 ng RNA was reverse transcribed into cDNA using the miRScript system (GenScript Co., Ltd., Nanjing, China) according to the manufacturer's protocols. qPCR was performed using SYBR Green qPCR Super mix (cat. no. CW0957M; Beijing CWBio) on a CFX96 Touch PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The thermocycling conditions were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec. All experiments were performed with triplicate. The relative expression of VEGF was normalized to β-actin using the 2^−ΔΔCq method (22). U6 was used as an endogenous control to analyze the expression of miR-204-5p. The primers for RT-qPCR were as follows: VEGF, forward, 5′-GAGTTAAACGAACGTACTTGCAGA-3′ and reverse, 5′-TCTAGTTCCCGAAACCCTGA-3′; β-actin, forward, 5′-TGGCTGGGGTGTTGAAGGTC-3′ and reverse, 5′-ATGGTGGGTATGGGTCAGAAGG-3′; miR-204-5p, forward, 5′-ACACTCCAGCTGGGTTCCCTTTGTCATCCTAT-3′ and reverse, 5′-CTCAACTGGTGTCGTGGA-3′; and U6, forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′.

Proteins were isolated from retinal tissues and rRECs using radioimmunoprecipitation assay buffer (Sigma-Aldrich; Thermo Fisher Scientific, Inc.). Protein concentration was determined by bicinchoninic acid kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocol. Equal amounts of protein (60 µg) were separated using SDS-PAGE on 12% gels and transferred to polyvinylidene difluoride membranes (Millipore; Merck KGaA). Following blocking with 5% non-fat milk for 1 h at room temperature, membranes were probed with rabbit anti-LC3B antibody (cat. no. ab51520; 1:3,000; Abcam) or anti-β-actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4°C. Then, membranes were washed with TBS plus Tween-20 (0.1%) and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (cat. no. sc-516078; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at 37°C. Target bands were developed with an enhanced chemiluminescence detection kit (Amersham; GE Healthcare, Chicago, IL, USA). Band densities were calculated using Quantity One software version 4.5.1 (Bio-Rad Laboratories, Inc.) and normalized to β-actin.

Retinal tissues were isolated from the different groups at 8 weeks following STZ injection, fixed with 10% freshly prepared ice-cold paraformaldehyde at 4°C overnight and cut into 5-µm sections. Sections were incubated with 5% bovine serum albumin (BSA; Beijing Solarbio Science & Technology Co., Ltd.) at 37°C for 1 h to block the nonspecific sites. Then, sections were incubated with rabbit polyclonal anti-LC3B antibody (1:1,000) for 2 h at room temperature. Following washing with PBS (3×), slides were probed with horseradish peroxidase-conjugated goat anti-rabbit antibodies for 2 h at room temperature. Immunoperoxidase binding was visualized by reaction with diaminobenzidine-H₂O₂ solution. Five non-overlapping fields were randomly captured by an inverted microscope at a magnification of ×400. LC3B-positive cells were stained in brown or dark yellow.

Data are presented as the mean ± standard deviation. All statistical analysis was performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). Statistical analyses were performed using one-way analysis of variance followed by Dunnett's post hoc tests. P<0.05 was considered to indicate a statistically significant difference.

In the present study, a diabetic rat model was generated by intraperitoneal injection of STZ; the expression of miR-204-5p in the diabetic rat model was then investigated. To determine whether the diabetic rat model was successfully constructed, glucose levels were analyzed at day 3 following STZ administration. Glucose levels in the STZ-treated group (23.01±3.71 mM) were significantly increased compared with the control group (4.62±1.51 mM; P<0.01; data not shown). This suggested that a diabetic rat model was successfully constructed. As presented in Fig. 1A, compared with the control group, relative miR-204-5p expression levels in retina tissues of the diabetes group were significantly upregulated (P<0.01). In addition, expression VEGF mRNA levels in the diabetes group were significantly increased compared with the control group (P<0.01; Fig. 1B). The results indicated that miR-204-5p and VEGF may be associated with the diabetes and contribute to the progression of DR.

LC3B-II levels are proportional to the number of autophagic vacuoles (7). To investigate the role of LC3B in the retina of diabetic rats, LC3B protein expression was determined via immunohistochemistry. As presented in Fig. 2A, compared with the control group, LC3B expression in the diabetic group was markedly reduced. These results revealed that the expression of LC3B was inhibited in retina tissues of diabetic rats.

Additionally, protein expression levels of LC3B were determined by western blotting. Compared with the control group, expression levels of LC3B-II in the diabetic group were significantly reduced (P<0.01; Fig. 2B and C). Furthermore, the ratio of LC3B-II/LC3B-I was significantly decreased in the diabetic group compared with the control (P<0.01; Fig. 2D). These data suggested that the expression of LC3B-II and the ratio of LC3B-II/LC3B-I may be suppressed during the development of DR.

To further investigate the mechanism by which miR-204-5p affects the progression of DR, anti-miR-204-5p and miR-204-5p mimic were administered to rats of the diabetes group. As presented in Fig. 3A, compared with the neg-miR group, anti-miR-204-5p treatment significantly suppressed miR-204-5p expression and miR-204-5p mimic treatment significantly upregulated miR-204-5p expression (P<0.01). The results suggested that anti-miR-204-5p and miR-204-5p mimic were functional in diabetic rats. In addition, compared with the neg-miR group, injection of anti-miR-204-5p into diabetic rats significantly enhanced LC3B-II expression and the ratio of LC3B-II/LC3B-I (P<0.01; Fig. 3B and C). In the presence of the miR-204-5p mimic these levels decreased compared with the neg-miR control (P<0.01). mRNA expression levels of VEGF were significantly upregulated in the anti-miR-204-5p and the mimic groups compared with the neg-miR control (P<0.01; Fig. 3D). Interestingly, miR-204-5p mimic treatment induced a larger increase in VEGF mRNA expression levels compared with the anti-miR-204-5p group.

In order to confirm the results in vitro, rRECs were isolated from diabetic rats and transfected with anti-miR-204-5p, miR-204-5p mimic or neg-miR. The purity of rRECs following isolation was determined via factor VII staining. The percentage of factor VII-positive cells was >97%, indicating that rRECs were successfully isolated from retina tissues of diabetic rats (Fig. 4A). As presented in Fig. 4B, compared with the neg-miR group, anti-miR-204-5p markedly increased the number of autophagic vacuoles and a reduction was observed in response to treatment with miR-204-5p mimic. The results of the transfection efficiency analysis are presented in Fig. 4C; it was determined that anti-miR-204-5p successfully inhibited and miR-204-5p mimic successfully enhanced miR-204-5p expression compared with the neg-miR control (P<0.01). Anti-miR-204-5p further significantly promoted LC3B-II expression and significantly increased the LC3B-II/LC3B-I ratio; miR-204-5p mimic exhibited opposing effects compared with the neg-miR control (all P<0.01; Fig. 4D and E). Additionally, anti-miR-204-5p and miR-204-5p mimic significantly upregulated the expression of VEGF compared with the neg-miR control (P<0.01; Fig. 4F). These results revealed that miR-204-5p may be involved in the development of DR by negatively regulating the expression of LC3B-II.