Cell culture
HEK293T cells from the American Type Culture Collection were used in the dual-luciferase reporter assay. HEK293T cells were cultured in the DMEM containing 10% fetal bovine serum by routine methods. Cells were passaged and cultured in fresh complete medium every three days.
Dual-luciferase reporter system assay
The bioinformatics prediction website (www.targetscan.org) predicted that there was a binding site between miR-874 and Rela (p65), which was then verified by dual-luciferase reporter system assay. The reporter plasmids containing target gene Rela (pmirGLO-Rela wt) and the mutant in the target gene (pmirGLO-Rela mut) were constructed, respectively. These two reporter plasmids were co-transfected with NC mimic or miR-874 mimic into HEK 293T cells, respectively. After transfection for 24 h, the dual-luciferase reporter assay was performed according to the instruction of dual-luciferase reporter assay kit (Promega). Relative luciferase activity = firefly luciferase activity / renilla luciferase activity.
Establishment of diabetes rat models
Streptozotocin (STZ) was used to induce diabetes rat models. Ninety male Sprague-Dawley rats (200-250 g, 8 weeks old, from the Laboratory Animal Center of Chongqing Medical University, China) were fed with standard food and water in the laboratory under specific pathogen free condition. Ten rats were randomly selected as the control group, and the rest were used to construct models. Citrate buffer solutions (pH 4.5) were used to prepare fresh STZ solutions. Single intraperitoneal injection of 60 mg/kg STZ solutions was performed in rats to induce diabetes. One week later, the rat with fasting blood glucose above 250 mg/dl was considered to be a successful model [19]. There were 71 successfully modeled rats. The protocol and procedures employed were ethically reviewed and approved by the Ethics Committee of Xiaogan Central Hospital (2017010) and in compliance with the statement of Association for Research in Vision and Ophthalmology for the care and use of laboratory animals in ophthalmology and vision studies.
Nine weeks after grouping, rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg). The left eyeball of all rats were detected by a color Doppler ultrasound to obtain retinal hemodynamic indexes and central artery hemorheology indexes and then the blood sample and retinal tissue or the whole eyeballs were collected for the subsequent experiments followed by cervical dislocation for euthanasia. The death of rats was determined by no respiration.
Grouping and disposing
Sixty successfully modeled rats were randomly selected and divided into 6 groups with 10 rats in each group and the rest were euthanatized by cervical dislocation under narcotism by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg). There were 7 groups in this study: control group (healthy rats), model group (diabetes rats injected with normal saline via caudal vein), NC (negative control) agomir group (diabetes rats injected with NC mimic via caudal vein), miR-874 agomir group (diabetes rats injected with miR-874 mimic via caudal vein), miR-874 anti-agomir group (diabetes rats injected with miR-874 inhibitor via caudal vein), EVP4593 group (diabetes rats injected with EVP4593 via caudal vein), and miR-874 anti-agomir + EVP4593 group (diabetes rats injected with miR-874 inhibitor and EVP4593). The details of grouping were shown in Table 1. EVP4593 was NF-κB signaling pathway antagonist. The above agentia at a concentration of 4.5 nM were injected into rats at the dose of 80 mg/kg via caudal vein, once every three days for 4 weeks [20]. Eight weeks later, rats were fasted for 8 h. Then blood was drawn via caudal vein to measure blood glucose by the One Touch II glucometer (USA). Rats were weighed. The experimental design was shown in Fig. 1.
Retinal hemodynamic indexes and central artery hemorheology indexes detection in diabetes rats
Nine weeks after grouping, the rat eyeball was examined by a color Doppler ultrasound, and hemodynamic indexes of the left eye such as end-diastolic velocity (EDV), peak systolic velocity (PSV) and central retinal vein (CRV) were detected in all rats. After the rats were fasted for 20 h, rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg) and then anticoagulated blood was drawn via the abdominal aorta. Plasma viscosity (PV), blood viscosity (BV) and erythrocyte sedimentation rate (ESR) at different shear rates were measured with a blood viscometer. The experiment was performed in triplicate.
Detection of number of retinal vascular endothelial cells and pericytes
The eyeballs of all rats were fixed. Retinal vascular digest preparations were performed. Number of retinal vascular endothelial cells (VEC) and capillary pericytes (IPC) was counted by using a microscope. All experiment was performed in triplicate.
Separation of retinal tissue
The eyeballs of rats were extirpated under aseptic conditions. The bulbar conjunction was removed. The cornea was separated at 1 mm from the posterior of corneoscleral limbus followed by the evisceration of crystalline lens and the removal of vitreous body under a stereomicroscope. The retina was isolated along the underpart of the retina, and the optic nerve was cut off. The retina was dissociated and cut into pieces.
qRT-PCR (Real-time fluorescence quantitative polymerase chain reaction)
Total RNA in the retinal tissue was extracted by the Trizol method (Invitrogen, Calsbad, CA, USA). After purity determination, the RNA was reversely transcribed into cDNA according to the instruction of TaqMan MicroRNA Assays Reverse Transcription primer (4427975, Applied Biosystems, USA) with reaction conditions of 37 °C for 30 min and 85 °C for 5 s. Primers were synthesized by the Wuhan Branch of Sangon Biotech (Shanghai) Co., Ltd., China and the sequences were listed in Table 2. Reaction condition of qRT-PCR was: pre-denaturation at 95 °C for 10 min followed by 40 circles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s and extending at 72 °C for 34 s. Reaction system of qRT-PCR was: 10 µl SYBR Premix Ex TaqTM II, 0.8 µl PCR forward primer (10 μM), 0.8 μl PCR reverse primer (10 μM), 0.4 μl ROX Reference Dye II, 2.0 μl cDNA templates, and 6.0 μl sterilized distilled water. U6 was used as the internal reference of miR-195, and GAPDH was the internal reference of CD40, RORyt, Foxp3, interleukin (IL) -17, IL-10, tumor necrosis factor (TNF)-α, IL-23 and IL-8. The reaction was performed on an ABI7500 quantitative PCR amplifier (7500, ABI, USA). 2-ΔΔCt showed the relative expression of target gene. The experiment was performed in triplicate.
Western blotting
RIPA (Beyotime Biotechnology Co., Ltd., China) was mixed with protease inhibitor and PMSF to lyse cells on the ice for 30 min. Protein concentration was measured by using BCA protein assay kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd., China). Protein was separated by SDS-PAGE for 2 h and transferred to PVDF membranes. The membrane was sealed with 5% milk for 2 h and incubated at 4 °C with the addition of primary antibodies including rabbit anti-human angiopoietin2 (Ang2) (1:2,500, ab155106, Abcam, USA), p65 (1:2,500, ab32536, Abcam, USA), vascular endothelial growth factor (VEGF) (1:2,500, ab1316, Abcam, USA) and GAPDH (1:2,500, ab9485, Abcam, USA). After the membrane was washed with tris buffered saline tween (TBST) three times, horse radish peroxidase-labeled IgG (1:10,000, ab6721, Abcam, USA) was added and incubated at room temperature for 1 h. Then the membrane was washed with TBST three times. Color development was carried out by electrogenerated chemiluminescence solutions. Relative expression of protein = gray value of target protein band / gray value of GAPDH band. The experiment was performed in triplicate.
Statistical analysis
Data analysis was performed by SPSS 11.5 software. The measurement data were expressed as mean ± standard deviation. Comparison among groups was performed by one-way ANOVA and post-hoc LSD-t test. P < 0.05 indicates that the difference is statistically significant.