Cell culture
The American Type Culture Collection provided the human retinal pigment epithelial cells ARPE-19 (ATCC, Manassas, VA, USA). ARPE-19 cells were cultured in DMEM/F-12 media (Gibco, New York, USA) supplemented with 10% FBS and 1% streptomycin/penicillin at 37°C in a humidified atmosphere with 95% air and 5% CO2. Then, the cells were exposed to 400 µM H2O2 (Sigma-Aldrich, St. Louis, MO, USA) and allowed to incubate for a duration of 6 hours. Lastly, H2O2-treated cells were used in the studies.
Transfection of miRNA inhibitors and mimics
MiR-6837-3p inhibitors and mimics were constructed by GeneChem (Shanghai, China). ARPE-19 cells after treated with H2O2 were inoculated at 1.5 × 105 cells/well in 12-well plates and then transfected with disordered miRNA (NC, 20 nM), miR-6837-3p inhibitor (20nM), and miR-6837-3p mimic (20 nM) using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's protocols.
Cell Counting Kit 8 (CCK8)
H2O2-treated ARPE-19 cells were inoculated into 96-well plates at a density of 1 × 104 cells /well and incubated for 24 hours to allow cells to attach to the surface. Next, for one to five days, a Cell Counting Kit 8 (CCK8; Beyotime Institute of Biotechnology, Beijing, China) solution was added. The OD values were measured at 570 nm using a microplate reader (M2009PR, Tecan infinite, Australia) at different time points.
Flow cytometry
The Annexin-V/PE Apoptosis Assay Kit (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) was used to detect apoptosis, and propidium iodide (PI) (P4170; Sigma, Co. St. Louis, MO, US) was used to detect cell cycle in accordance with the manufacturer's protocol. Finally, data were collected by a the FACScan flow cytometer (BD Biosciences, New Jersey, USA) and analyzed using CellQuest software (version 7.6.1; Flow Jo LLC).
Wound-healing assay
H2O2-treated ARPE-19 cells were transfected with inhibitors or mimics for 24 hours and then grown into 6-well plates. When 80% confluence was reached, we scraped the cell monolayer with a sterile pipette. After washing three times with PBS, the cells were cultured in FBS-free medium. Images shown are from 2 time points: 0 and 24 hours, and demonstrate wound closure as measured using ImageJ.
Luciferase reporter assays
Wild-type E2F6 (E2F6 3’UTR-WT) and 3'-UTR mutant plasmid of E2F6 (E2F6 3’UTR-MUT) were synthesized by GeneChem (Shanghai, China), and then, these vectors were co-transfected with NC mimics or miR-6837-3p mimics into the cells using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA). Forty-eight hours after transfection, luciferase activity was monitored using a dual luciferase reporter system (Promega, Madison, WI 53711 USA).
Immunofluorescence
H2O2-treated ARPE-19 cells were transfected with inhibitors or mimics were cultured in 6-well plates and fixed in 4% formaldehyde for 30 min and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. After blocking the frozen cells for 30 minutes at room temperature with 5% BSA, they were treated with antibodies targeting E2F6 (1:100 dilution; #ab289963, Abcam, Cambridge, UK) and Caspase3 (1:200 dilution; #ab208161, Abcam, Cambridge, UK) for an overnight period at 4°C. After one hour at 37°C incubation with the secondary antibody Goat Anti-mouse-FITC (#S0007, Affinity, Changzhou, China) or Goat Anti-rabbit-Fluor594 (#S0006, Affinity, Changzhou, China), the cells were counterstained for ten minutes with DAPI (Sigma-Aldrich, St. Louis, MO, USA). Next, the cells were seen using a Carl Zeiss, Germany, LSM710 confocal microscope.
RT-qPCR
TRIzol reagent (Thermo Fisher, Waltham, MA, USA) was used to extract the total RNA from H2O2-treated ARPE-19 cells, and M-MLV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) was used to reverse transcribe the RNA into cDNA. SYBR Green Master Mix (Vazyme, Nanjing, Jiangsu, China) was used for the qPCR. Quantification of gene expression was carried out using the 2 − ΔΔCt technique. MiR-6837-3p was normalized to U6. Primer sequences: miR-6837-3p (Forward: 5′-GCAGGGTCCGAGGTATTC-3′, Reverse: 5′-CGTCCTTCACTGTGACTCTG-3′); U6: (Forward: 5′-CTCGCTTCGGCAGCACA-3′, Reverse: 5′-AACGCTTCACGAATTTGCGT-3′).
Western blot analysis
Total proteins were extracted from cells with cell lysis buffer (RIPA, Beyotime, Shanghai, China) containing protease inhibitors (PMSF, Boster, Wuhan, China). Utilizing a BCA assay kit (Thermo Fisher, Waltham, MA, USA), the protein content was determined. Proteins were electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF, Millipore, MA, USA) membranes. The membranes were then blocked for 1 hour at room temperature with 5% skim milk, and then incubated with primary antibodies for an additional night at 4°C. Information on the primary antibodies used in the study is as follows: anti-E2F6 (1:1000, # ab289963, Abcam, Cambridge, UK), anti-Caspase3 (1:2000, # ab184787, Abcam, Cambridge, UK), anti-Caspase9 (1:2000, # ab202068, Abcam, Cambridge, UK), and anti-GAPDH (1:3000, #AF7021, Affinity, Changzhou, China). The next day, membranes were incubated for 1 hour at room temperature with Goat Anti-Rabbit IgG secondary antibody (1:1000, #S0001, Affinity, Changzhou, China). The images were then obtained by scanning the blots. With the aid of a very sensitive ECL chemiluminescence detection kit, the tagged protein bands were examined.
Statistical analysis
All data were analyzed using GraphPad Prism software 8.0 (San Diego, CA, USA). All experiments were repeated 3 times and presented as the mean ± standard deviation (SD). Quantitative variables were analyzed by the Student's t-test between groups. Then, p < 0.05 was considered significant.