Cell culture and treatment
The ARPE-19 cells (ATCC, Rockefeller, Maryland, USA) were cultured in DMEM/F-12 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies, Carlsbad, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St Louis, MO, USA) at 37°C in humidified air with 5% CO2. ARPE-19 cells were exposed to a clinical concentration (0.25 mg/ml) of BEV (Roche Diagnostics, GmbH, Germany) for 2 days. In some experiments, 10 μM of SB431542 (Tocris Bioscience, Minneapolis, Minnesota, USA) and 2μM of SIS3 (Aladdin, Shanghai, China) were added in the culture medium.
Cell transfection
The ARPE-19 cells divided into several groups: the negative control (NC) group (ARPE-19 cells were treated with BEV and transfected with negative control sequence of miR-21 inhibitor or mimic); miR-21 inhibitor group (ARPE-19 cells were transfected with 50 nM of miR-21 inhibitor (RiboBio Biotechnology, Guangzhou, China) one day before BEV treatment); miR-21 mimic group (ARPE-19 cells were transfected with 50 nM of miR-21 mimic (RiboBio Biotechnology) one day before BEV treatment).
qRT-PCR assay
The quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to detect the expression of miR-21 in ARPE-19 cells. Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) from ARPE-19 cells and the level of miR-21 was determined using specific primers (RiboBio Biotechnology, Guangzhou, China). U6 was used for normalization. qRT-PCR was performed using the Qiagen Rotor-Gene quantitative PCR system according to the manufacturer's protocol (Vazyme Biotech, Nanjing, China). Reaction conditions: pre-change for 10 seconds at 95°C, 5 seconds at 95°C, and 20 seconds at 60°C for 40 cycles. The expression level of the gene is represented by 2-ΔΔCt (Ct represents a cycle threshold).
Western blot analysis
Total protein was extracted from lysis of ARPE-19 cells in a radioimmunoprecipitation assay (RIPA) buffer containing a mixture of protease inhibitors and 50 μg of total protein per sample were loaded and separated on 10% SDS-PAGE, and then wet-transferred using PVDF membranes (Rugby WAR, UK). The membranes were blocked in 5% skimmed milk powder in TBST and incubated overnight with specific primary antibodies of E-cadherin (1:1000; Abcam), α-SMA (1:1000; Abcam), Snail (1:1000; Cell Signaling Technology, Trask Lane, Danvers, USA), TGFβ1 (1:1000; Cell Signaling Technology), Phospho-smad2/3 (1:1000; Cell Signaling Technology) and total-smad2/3 (1:1000; Cell Signaling Technology). On the second day, horseradish peroxidase-labeled secondary antibody (goat anti-rabbit IgG at a concentration of 1:2000, ZSGB-BIO, Beijing, China) was added dropwise for 1 hour at room temperature. After the end of the immunological reaction, the cells were illuminated and developed with a western blot chemiluminescence reagent (Invitrogen, Carlsbad, CA, USA). Images were captured using a western blot imaging system (Bio-rad ChemiDoc Touch, USA) and were analyzed using Image J version software (NIH, Bethesda, MD, USA).
Statistical analysis
Statistical analysis was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA), and the data were expressed as mean ± standard deviation. The data between two groups were analyzed by unpaired t-test. One-way ANOVA test was used to determine the statistical difference among three groups. Values of p<0.05 were considered significant.