2.1 Capsular injury model in rats.
1 month old Wistar rats were purchased from SLAC Laboratory Animal Co.,Ltd (Shanghai, China). Animal care and experimental protocols were complied with guidelines of Animal Ethics Committee of Harbin Medical University (number: sydwgzr2020-11). After anesthetized generally with pentobarbital sodium (70 mg/kg) and topically with dicaine eye drop, a 1 mm incision was made at the transparent cornea at 12 O’clock’ and filled with hyaluronic acid to maintain the state of the anterior chamber. After cutting off the anterior capsule, BSS solution was injected into the capsule to completely separate the lens cortex from the capsule. Then sutured the corneal incision. The occurrence of PCO was observed by slit lamp microscope at 0, 3, 7, 14, 21 and 28 days after operation25. All the animals were intraperitoneally injected with 3% pentobarbital sodium and were euthanized by excessive anesthesia with a dose of 90 mL/kg. Then the eyes were enucleated and posterior capsular tissue of the lens were removed for the experiments.
2.2 Cell culture
Human lens epithelial cells SRA10/04 was purchased from ATCC (Manassas, VA, USA). The cells were cultured with complete medium including 89% DMEM and 10% FBS (Biological Industries, Beit-Haemek, Israel) and maintained in incubator with 37°C and 5% of CO2 saturated humidity.
The final concentration of TGF-β2 (PeproTech, Rocky Hill, NJ, USA) was 10 ng/ml. Before being collected for further analysis, the cells were cultured in the medium containing TGF-β2 for 48 hours.
2.3 Western blot
Cells were lysed with RIPA buffer and 45 ug proteins were run on a 8% SDS-polyacrylamide gel then transferred to PVDF membrane. After 1 hour of blocking with 5% skim milk, the membranes were incubated with primary antibodies overnight at 4℃. The corresponding secondary antibodies were incubated at room temperature for 1 hour the next day. Primary antibodiy against IGF-1 (DF6096, 1:500), Vimentin (AF7013, 1:2000), E-cadherin (AF0131, 1:500) and β-actin (AF7018, 1:2000) were purchased from Affinity Biosciences (Jiangsu, China), with β-actin as an internal control. Odyssey Infrared Imaging System (Odyssey CLx, Biosciences, USA) was used to detect immunoreactivity.
2.4 qRT-PCR.
Trizol reagent was used to extract RNA from cells or tissues. NanoDrop 8000(Thermo Scientific, Waltham, MA, USA) was used to detect the concentration and purity of RNA. The single-stranded cDNAs were synthesized from 1 µg of RNA. The expression of mRNAs and miRNAs were quantified by RT-PCR with SYBR Green I (Thermo Fisher Scientific, Inc). Primer sequence of human genes:
GAPDH:
F: 5’-ACCACAGTCCATGCCATCAC-3’
R: 5’-TCCACCACCCT GTTGCTGTA-3’;
miR-3666:
F: 5’-ACGAGACGACGACAGAC-3’
R: 5’-CAGTGCAAGTGTAGATGCCGA-3’;
U6:
F: 5’-GCUUCGGCAGCACAUAUACUAAAAU-3’
R: 5’-CGCUUCACGAAUUUGCGUGUCAU-3’ ;
IGF-1:
F: 5’-CAGCAGTCTTCCAACCCAAT-3’
R: 5’-GCTGACTTGGCAGGCTTGAG-3’ ;
CDH1:
F: 5’-CTGAGAACGAGGCTAACG-3’
R: 5’-CCACCATCATCATTCAATATG-3’ ;
N-cadherin:
F: 5’-ACACTGGTGGCACTACTAAG-3’ and R:5’-TACACAATACAGAGGCAAAG-3’;
Primer sequence of rat IGF-1,
F: 5’-CTTTACCAGCTCGGCCACA-3’
R: 5’-TTGGTCCACACACGAACTGAAG-3’.
2.5 miRNA and plasmid transfection.
The LECs were plated until the cell density reached 80% confluency of dishes to transfect. The miRNAs/inhibitors/plasmids were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). MiR-3666 mimics/inhibitor were bought from RiboBio (Guangzhou, China) and plasmid of IGF-1 or short hairpinRNA of IGF-1 (sh-IGF-1) were constructed by Genechem (Shanghai, China).
2.6 EdU assay
Using EdU Cell Proliferation Kit (RiboBio, Guangzhou, China) to test proliferation. LECs were seeded in 24-well plates. After miRNA or plasmid treansfection for 48h, cells were maintained with 200uL 50 uM EdU and for 2 h. Apollo Dye Solution (red) were used to stain proliferating cells, nucleic acids were stained with DAPI (blue) according to the protocols26 and then photographed.
2.7 Wound-healing assay
To test the cell migration ability of LECs, wound healing assay was performed. LECs were plated and cultured with FBS-free medium in 6-well plates until the cells formed a confluent monolayer, then scratched using a 100 µL pipette tip. The scratch wounds were captured using microscopy at 0, 24h. The wound area was analyzed by image J.
2.8 Luciferase reporter assays
To verified if IGF-1 was a target of miR-3666, the 3′-UTR of IGF-1 including the predicted binding sites wild (wt) or mutated (mut) binding sites were inserted into pmirGLO vector. The reporter plasmids of IGF-1 were co-transfected with miR-3666 mimics or miR-NC. Luciferase activities were measured by dual luciferase reporter assay kit (Promega, USA) after 24 h.
2.9 Matrigel transwell assay
24-well matrigel transwell (Corning, USA) were used to investigated cell invasion. 2 × 105 LECs were seeded on the cell culture insert precoated with 1 µg/µL Matrigel (BD Biosciences, USA). Medium with FBS was used to stimulate invasion in the bottom of wells. After 48 h, the invasion cells were stained with a 0.1% crystal violet solution. The number of invading cells were counted under microscope.
2.10 Statistical analysis
Data were shown as mean ± SEM. Unpaired Student’s t-test or one-way ANOVA was used to compare the groups. P < 0.05 was considered significant