Cell lines and cell culture
HRMECs were purchased from American type culture collection (ATCC). HRMECs were cultured in Dulbecco's modified eagle's medium (DMEM) (Sigma-Aldrich, USA) supplemented with 1% penicillin/streptomycin (100 mg/L, Gibco, USA) and 10% heat-inactivated fetal bovine serum (FBS) (Gibco, USA) at 37 °C in 5% CO2 atmosphere. For normal glucose and high glucose conditions, 5 mM and 33 mM D-glucose (Gibco, USA) were added to the medium for 48 h, respectively.
All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai General Hospital and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. C57BL/6 male mice were purchased from Shanghai Model Organisms Center. The animals were housed in cages with free access to regular diet and water in a room at 22 ± 1 °C on a 12 h light/dark cycle. When the mice reached 20–25 g body weight (∼2 months of age), they were randomly assigned into diabetic or nondiabetic group. Diabetes was induced by five sequential daily intraperitoneal injections of a freshly prepared solution of streptozotocin in citrate buffer (pH 4.5) at 45 mg/kg body weight. Mice with random blood glucose levels ≥ 16.7 mmol/L at 2 weeks post-STZ were assigned to the diabetes group and the diabetes duration commenced. The animals had free access to food and water. Retinal tissues were harvested at 9 months of diabetes for protein extraction, RNA extraction, and retinal histopathology. Fasting blood glucose levels were determined repeatedly prior to the 3-month assessment.
For subretinal injection, adeno-associated virus (AAV) vector containing sh-tiRNA-Val under the control of chimeric CMV/chicken β-actin promoter was constructed. The vectors were administered via subretinal injection two weeks before STZ induction of diabetes. C57BL/6 male mice were anesthetized and subretinally injected with 1μL solution containing 1011 particles of sh-tiRNA-Val AAV, as previously described . The solution was injected only in one eye for each animal, while the contralateral eye was used as a control. Retinal tissues were harvested after 9 months of diabetes.
tiRNA-Val mimics, tiRNA-Val inhibitors, and corresponding negative controls were purchased from Sangon Biotech (Shanghai, China). Lipofectamine 3000 transfection reagent (Invitrogen, USA) was used for cell transfection according to the manufacturer's instructions. The final concentrations of tiRNA-Val mimics and tiRNA-Val inhibitors were 50 nM, respectively.
Cell proliferation assay
Cell viability was assessed using CCK-8 assay (Cell Counting Kit-8, Sigma-Aldrich, USA) according to the manufacturer’s instructions. Briefly, 5 × 103 cells/well were seeded into 96-well plates. Proliferative activity was determined at the end of different experimental periods (24 h, 48 h, 72 h, and 96 h). When the medium changed from red to yellow, the absorbance value at a wavelength of 450 nm was detected using an enzyme-linked immunosorbent assay reader (Thermo Fisher Scientific, USA). The experiment was performed at least three times with similar results.
Transwell migration assay
The migratory ability of HRMECs was assessed using 24-well transwell migration chambers (8 μm size, Corning, USA). Briefly, 5 × 104 cells/well were resuspended in 200 μL serum-free DMEM and inoculated evenly into the inner chambers. The bottom chambers were replenished with 500 μL of DMEM containing 20% FBS as an attractant. After 24 h, the cells migrated to the lower chamber through the hole, fixed with 4% paraformaldehyde, and then stained with 0.1% crystal violet.
Cell lysates or mouse tissues were prepared using 1 × cell lysis buffer (Cell Signaling Technology, USA) with 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich, USA). Protein lysate of 10–20 μg was run on 10–15% SDS-PAGE gel and transferred toa PVDF membrane (Roche, USA). The membrane was incubated for 60 min at room temperature in 5% BSA solution. The following antibodies were used for the detection of protein expression: actin (1:1,000,Sigma, USA), angiogenin (Ang) (1:1,000,Abcam, USA), VEGF (1:1,000,Thermo Fisher Scientific, USA),ZO-1 (1:1,000,Thermo Fisher Scientific, USA), ICAM-1 (1:1,000,Abcam, USA), Sirt1 (1:1,000,Cell Signaling Technology, USA), and Hif-1α (1:1,000,Cell Signaling Technology, USA). Anti-rabbit and anti-mouse peroxidase-conjugated secondary antibodies (1:2,000,Cell Signaling Technology, USA) were purchased from Jackson Immunoresearch, and the signal was visualized using western blotting luminol reagent (Thermo Fisher Scientific, USA).
Quantification of mRNA by RT-qPCR
Total RNA was isolated from cultured cells or mouse tissues using TRIzol reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. For mRNA quantification, cDNA was synthesized using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, USA) with random primers. RT-qPCR was performed using SYBR Green method. The primers used for amplification are listed in Additional file 2: Table S1, and each experiment was repeated at least three times independently. The mRNA expression levels were calculated using β-actin as an internal control.
Quantification of tiRNA by TaqMan RT-qPCR
TaqMan RT-qPCR for specific quantification of tiRNA was performed as previously described. Briefly, total RNA was treated with T4 PNK (New England Biolabs, UK), followed by ligation to 3'-RNA adapter using T4 RNA ligase. Ligated RNA was then subjected to TaqMan RT-qPCR using SuperScript IV Reverse Transcriptase, 200 nM of TaqMan probe targeting the boundary of target RNA and 3'-adapter, and specific forward and reverse primers. The expression of tiRNA was calculated using 5S RNA as an internal control. The sequences of the TaqMan probes and primers are listed in Additional file 2: Table S2.
RNA cleavage reaction in vitro
RNA cleavage was performed as previously described . Briefly, the incubation mixtures contained 20 μg of total RNA extracted from HRMEC, 30 mM HEPES, pH 6.8, 30 mM NaCl, 0.001% BSA, and recombinant human angiogenin protein (R&D Systems, USA) at concentrations of 0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM, and 2.0 μM. Incubation was performed at 37 °C for 30 min. The cleaved products were recovered through phenol–chloroform extraction and ethanol precipitation. Then, the products were analyzed through northern blotting.
Northern blotting for specific detection of small RNA was performed as previously described . Briefly, total RNA was separated using 15% urea PAGE. Gels were stained with SYBR Gold nucleic acid gel stain (Thermo Fisher Scientific, USA) and immediately imaged and transferred to positively charged nylon membranes (Roche, Switzerland). Subsequently, the membranes were air-dried and UV-crosslinked. The membranes were pre-hybridized with DIG Easy Hyb buffer (Roche, Switzerland) for at least 1 h at 45 °C. For the detection of specific small RNAs, the membranes were incubated overnight at 45 °C with 10 nM 3'-DIG–labeled oligonucleotide probes synthesized by Sangon Biotech (Shanghai, China), as shown in Additional file 2: Table S3. The membranes were washed twice with low stringent buffer (2 × SSC with 0.1% (w/v) SDS) at 37 °C for 15 min each, then rinsed twice with high stringent buffer (0.1 × SSC with 0.1% (w/v) SDS) at 37 °C for 5 min each, and finally rinsed in washing buffer (1 × SSC) for 10 min. Following the washes, the membranes were transferred onto 1 × blocking buffer (Roche) and incubated at room temperature for 2–3 h, after which DIG antibody (Roche) was added to the blocking buffer at a ratio of 1:10,000 and incubated for an additional 1/2 min at room temperature. The membranes were then washed four times in DIG washing buffer (1 × maleic acid buffer, 0.3% Tween-20) for 15 min each, rinsed in DIG detection buffer (0.1 M Tris–HCl, 0.1 M NaCl, pH 9.5) for 5 min, and then coated with CSPD ready-to-use reagent (Roche, Switzerland). The membranes were incubated in the dark with CSPD reagent for 15 min at 37 °C before imaging using the Carestream imaging system.
HEK293T cells in a 24-well plate were co-transfected with pSIF-GFP or the indicated plasmids expressing tiRNA (0.8 μg/well), pRL-Sirt1-3′ UTR (pRL-TK vector containing Sirt1 3′UTR) or pRLSirt1- 3′UTRm (pRL-TK vector containing mutant Sirt1 3′UTR) (0.1 μg/well), and pSV40-β-gal (Promega, Madison, WI, USA) (0.1 μg/well) using lipofectamine 3000. HERMEC cells in a 24-well plate were co-transfected with the indicated tiRNA mimics, pRL-Sirt1-3′UTR (0.1 μg/well), and pSV40-β-gal (0.1 μg/well) using lipofectamine 3000. After transfection for 72 h, the cells were harvested for luciferase assay as previously described .
Quantitative data are represented as mean ± SD. All images are representative of the studies with three to nine animals per group. Paired Student's t-test was used to assess the significant difference between the two groups. Statistical significance was set at p ≤ 0.05.