Cell lines and cell culture
HTM cells were acquired from cadaver eyes with the approval of the Ethics Committee of Nanjing Tongren Hospital (Jiangsu, China). HTM cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM; USA) containing 10% fetal bovine serum (FBS; Invitrogen, USA), 100 U/ml penicillin (Sigma-Aldrich, USA) and 100 μg/ml streptomycin (Sigma-Aldrich, USA), and then stored in the humidified atmosphere with 5% CO2 at 37℃.
The cultured HTM cells were treated with disparate concentrations of H2O2 (0 – 300 μM) to establish an in vitro oxidative cell damage model. Non-disposed cells acted as a control.
For overexpression of SNAI2, the SNAI2 sequence was synthesized and inserted into the pcDNA3.1 (Invitrogen, U.S.A.) plasmid to produce the pcDNA3.1/SNAI2. For knockdown of SNHG3, SNAI2 or ELAVL2, short hairpin RNA (shRNA) specifically targeting SNHG3, SNAI2 or ELAVL2 were devised and synthesized by GenePharma (Shanghai, China). Lipofectamine 2000 (Invitrogen, U.S.A.) was applied for cell transfection in line with manufacturer’s recommendations.
The viability of HTM cells were detected by using Cell Counting Kit-8 (CCK-8). The transfected cells with 1×104 cells per well were planted in 96-well plates. After incubation for 48 h, CCK-8 solution (10 μl) was supplemented into each well. After that, the incubation continued for 4 h. Cell viability was visualized using a microplate reader (EL340; BioTek Instruments, Hopkinton, MA) at a wavelength of 450 nm.
Flow cytometry analysis
The flow cytometry was conducted to assess HTM cell apoptosis with Annexin V Kit (Beyotime, China) with reference to protocols stipulated by the manufacturer. Cells transfected with indicated plasmid or negative control were collected 48 h later and washed twice with PBS. Subsequently, cells (1 × 106 cells/mL) were double stained by propidium iodide (PI) and Annexin V-FITC. The CELLQUEST program (Becton Dickinson, USA) was used to record cells.
Transfected cells were lysed applying RIPA buffer (Thermo, USA), and cultured for 15 min at 4°C. Then, the lysate was centrifuged, and the concentrations of the proteins were measured using an BCA Protein Assay Kit (Thermo, USA). The proteins were isolated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and moved onto PVDF membranes (Thermo, USA). Then, 5% skim milk was used to block the membranes for 1 h. Next, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies were obtained from Abcam company (Shanghai, China): SNAI2 (ab51772), ELAVL2 (ab72603), collagen Ⅰ (ab34710), fibronectin (ab2413), collagen III (ab7778), MMP3 (ab52915), MMP9 (ab76003) and GAPDH (ab8245). Subsequently, secondary antibody was used to be incubated with the membranes for 2 h at room temperature. Finally, an enhanced chemiluminescence kit (GE Healthcare, Chicago, IL) was adopted to observe the signals.
Real-time quantitative polymerase chain reaction (RT-qPCR)
TRIzol reagent (ThermoFisher Scientific Invitrogen, USA) was employed to extract total RNA. Then, total RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA). RT-qPCR analysis was performed using a SYBR Premix Ex Taq II kit (Cat. #RR820A, TaKaRa) on the 7500 Real-Time PCR Systems (Applied Biosystems). U6 served as an endogenous control for miRNAs. The detection results for mRNAs were normalized to GAPDH. Expression fold changes were calculated adopting the 2-ΔΔCt method.
Subcellular fractionation assay
Total RNA was extracted applying a DNA/RNA Isolation Kit (DP422, Tiangen Biotech, Beijing, China) in line with the instruction. The nuclear and cytoplasmic fractions were separated applying NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Total RNA was isolated from the nuclear and cytoplasmic fractions using Trizol (Invitrogen).
RNA pull down assay
The biotinylated RNAs were cultured with cell lysates, and then cultured with streptavidin beads (Invitrogen). Subsequently, the RNAs in complexes captured by streptavidin beads were washed, purified and assessed by western blot analysis.
RNA immunoprecipitation (RIP) assay
The RIP assay was carried out with a Millipore EZ-Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore, Bedford, MA, USA). The precipitated RNAs were evaluated using RT-qPCR. Antibodies against ELAVL2 and IgG were used. IgG served as the negative control.
Data analysis was conducted using SPSS 23.0 (IBM SPSS, Chicago, IL, USA). All data are exhibited as the means ± SD. Statistical significance among more than 2 groups was calculated using one-way ANOVA and Tukey’s post-hoc test. Difference between 2 groups was evaluated using Student’s t-test. P < 0.05 was regarded to be statistically significant.