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4 protocols using mir 18a mimic

1

Overexpression and Inhibition of miRNAs

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The transfection was performed using Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer. The transfected cells were cultured for 48 h before analysis. The CASC2 overexpression vector (pcDNA3.1-CASC2), empty vector (pcDNA3.1), miR-18a inhibitor, miR-21 inhibitor, inhibitor control, miR-18a mimics, miR-21 mimics and mimic control were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China).
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2

UV Radiation-Induced Corneal Cell Damage

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Damage caused by ultraviolet radiation is an important factor in corneal injury.26 (link) HCECs (CP-H128; Procell, Austin, TX, USA) were grown in DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA) containing 6% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 7 g/mL fetal bovine insulin (Sigma-Aldrich), and 7 ng/mL recombinant human epithelial growth factor (Sigma- Aldrich) at 37°C in a 5% CO2 (link) atmosphere. After referring to relevant literature,27 (link) HCECs were exposed to UV light (5.73 W/m2 (link)) at a distance of 30 cm for 5 min at 37°C in a 5% CO2 (link) atmosphere in a special apparatus. The UV intensity used for the control group was 0.1719 J/cm². An miR-18 inhibitor, negative control (NC), and miR-18a mimics were purchased from GenePharma (Suzhou, China). HCECs were seeded into the wells of a 6-well plate (1 . 105 cells/well). The cells were then treated with SH and transfected for 48 h with miR-18a mimics by using Lipofectamine 3000 (Invitrogen) as described in the manufacturer’s instructions.
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3

Knockdown and Overexpression of WDFY3-AS2 and PTEN

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The siRNA for WDFY3-AS2 (si-WDFY3-AS2) or PTEN (si-PTEN) was used to knock down WDFY3-AS2 or PTEN. WDFY3-AS2 plasmid was subcloned into the pcDNA3.1 vector to overexpress WDFY3-AS2 (pcDNA3.1-WDFY3-AS2). MiR-18a mimic, miR-18a inhibitor, and corresponding negative controls (NC-mimic and NC-inhibitor) were purchased from GenePharma Company (Shanghai, China). The transfections were performed using by Lipofectamine 2000 reagent (Invitrogen) following the instructions of the manufacturer.
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4

Evaluating miR-18a Modulation in HUVECs

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HUVECs were obtained from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultivated in Dulbecco's Modified Eagle Medium (Life Technologies/Thermo Fisher Scientific, Waltham, MA) with 10% fetal bovine serum (Invitrogen/Thermo Fisher Scientific, Waltham, MA) and incubated at 37°C in a 5% CO 2 humidified incubator. Total cells were separated into 3 groups: (1) miR-18a mimic group (cells transfected with miR-18a mimic); (2) miR-18a inhibitor group (cells transfected with miR-18a inhibitor); and (3) control group. Transfection efficiency was monitored by RT-qPCR. miR-18a mimic and miR-18a inhibitor were synthesized by Genepharma (Shanghai, China). Cells between passage 2 and passage 5 were used in this study.
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