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 assay
The binding site of miR-874 and Rela (p65) was analyzed on a bioinformatics prediction website (www.targetscan.org). Then a dual-luciferase reporter system was used to verify the target relationship between miR-874 and Rela. The gene vector of target gene Rela in the dual-luciferase reporter system and the mutant in the binding site of miR-874 and Rela were constructed: pmirGLO-Rela wt and pmirGLO-Rela mut. These two reporter plasmids and NC mimic or miR-874 mimic were co-transfected into HEK 293T cells respectively. After 24 h cell transfection, the dual-luciferase reporter assay was performed. Cells were lysed and centrifuged at 12,000 rpm for 1 min. The sediment was discarded, and the supernatant was collected. Luciferase activity was measured according to the instruction of dual-luciferase reporter assay kit (Promega). Operating steps were as follows. The lysed cells were pipetted into Eppendorf tubes. Every 10 μl cells were added with 100 μl firefly luciferase working solutions to measure firefly luciferase activity followed by the addition of 100 μl renilla luciferase working solutions to detect renilla luciferase activity. 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 . 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. All rats were anesthetized to collect the eyeballs after modeling followed by euthanasia.
Grouping and disposing
Sixty successfully modeled rats were randomly selected and divided into 6 groups with 10 rats in each group. There were 7 groups in this study: control group (healthy rats), model group (model rats, injection of normal saline via caudal vein), negative control (NC) agomir group (model rats, injection of overexpressed NC vector via caudal vein), miR-874 agomir group (model rats, injection of miR-874 mimic via caudal vein), miR-874 anti-agomir group (model rats, injection of miR-874 inhibitor via caudal vein), EVP4593 group (model rats, injection of NF-κB signaling pathway antagonist EVP4593 via caudal vein), and miR-874 anti-agomir + EVP4593 group (model rats, injection of miR-874 inhibitor and EVP4593). The details of grouping were shown in Table 1. The above mimics and antagonists of 80 mg/kg at a concentration of 4.5 nM were injected into rats via caudal vein, once every three days for 4 weeks . Eight weeks later, rats were fasted for 8 h. Then blood was drawn via caudal vein, and blood glucose was measured with the One Touch II glucometer (USA). Rats were weighed. The experiment was performed in triplicate. 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 detected 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. Anticoagulated blood was drawn via the abdominal aorta after the rats were fasted for 20 h. Plasma viscosity (PV), blood viscosity (BV) and erythrocyte sedimentation rate (ESR) at different shear rates were measured with a blood viscometer.
Detection of number of retinal vascular endothelial cells and pericytes
The eyeballs of all rats were extirpated and fixed. Retinal vascular digest preparations were performed. Number of retinal vascular endothelial cells (VEC) and capillary pericytes (IPC) was counted by using a microscope.
Separation of retinal tissue
General anesthesia was performed on rats by intraperitoneal injection of 1% pentobarbital sodium before the separation of retina. Surface anesthesia was performed on the eye by using oxybuprocaine hydrochloride eye drops. Then 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.
Total RNA in the retina was extracted by the Trizol method (Invitrogen, Calsbad, CA, USA). The ratios of A260/A230 and A260/A280 were measured by a nanodrop2000 micro-ultraviolet spectrophotometer (1011U, nanodrop, USA) to determine the concentration and purity of total RNA. RNA was reversely transcribed into cDNA according to the instruction of TaqMan MicroRNA Assays Reverse Transcription primer (4427975, Applied Biosystems, USA). cDNAs were diluted to 50 ng/μl, for 2 μl every time, and amplification reaction solutions were 25 μl. Reverse transcription reaction conditions: reacting at 37 °C for 30 min and at 85 °C for 5 s. Primers of CCL22 were synthesized by the Wuhan Branch of Sangon Biotech (Shanghai) Co., Ltd., China (Table 2). Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was performed by using an ABI7500 quantitative PCR amplifier (7500, ABI, USA). Reaction conditions: 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. Fluorescence quantitative PCR amplification solutions were 20 μl, including 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 to analyze the relative expression of miR-195, and GAPDH was used as the internal reference to analyze the relative expressions of CD40, RORyt, Foxp3, interleukin (IL) -17, IL-10, tumor necrosis factor (TNF)-α, IL-23 and IL-8. 2-ΔΔCt showed the relative expression of target gene. ΔΔCt = ΔCtexperimental group - ΔCtGAPDH. ΔCt = Cttarget gene - Ctinternal reference. Ct represented amplification circles as the real-time fluorescence intensity of the reaction reached the threshold value. The experiment was performed in triplicate.
p65 expression was detected by Western blotting. RIPA (Beyotime Biotechnology Co., Ltd., China) was mixed with protease inhibitor and PMSF to lyse cells on the ice for 30 min. Cells were scraped into 1.5 ml Eppendorf tubes with a cell scraper and centrifuged at 10,000 rpm and 4 °C for 15 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 polyclonal antibodies 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 cells were 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 cells were 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.
SPSS 11.5 software was used to analyze the data. The measurement data were expressed as mean ± standard deviation. Comparison among groups was performed by one-way ANOVA in conjunction with post-hoc LSD-t test. P < 0.05 was considered to be statistically significant.