Primary mGCs were isolated as described based on our previous study [21 (link)]. mGCs were grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12; Sigma-Aldrich, CA) supplemented with 10% inactivated fetal bovine serum (FBS; Gibco BRL, CA), penicillin (100 U/mL), and streptomycin (100 U/mL) (Life Technologies, CA) at 37°C in a humidified atmosphere with 5% CO2. The media were changed every 48 h. The 2- or 3-passage mGCs were selected for subsequent experiments.
A siRNA directly targeting MALAT1 (si-MALAT1; 5′-GTGAATGAGTGATAAGTAA-3′) and appropriate nontargeting siRNA (si-NC; 5′-TTCTCCGAACGTGTCACGT-3′) were bought from Geneseed Biotech Co. (Guangzhou, China). In addition, GenePharma Co. Ltd. (Shanghai, China) provided miR-205 mimics (5′-UCCUUCAUUCCACCGGAGUCUG-3′), negative control mimics (miR-NC; 5′-GGUCCGUCCGUAAUUAUCCUCC-3′), and a miR-205 inhibitor (anti-miR-205; 5′-CAGACUCCGGUGGAAUGAAGGA-3′) as well as its negative control (anti-miR-NC; 5′-CAGACUCCGGUGGAAUGAAGGA-3′). The CREB1 overexpression plasmid came from our lab. mGCs (5 × 103 cells/well) underwent transient transfection with the abovementioned constructs (100 nM siRNAs, mimics and inhibitors) using Lipofectamine 3000 (Invitrogen, CA, USA) based on provided protocols, with the efficiency of transfection being assessed at 48 h following transfection by quantitative real-time PCR (qRT-PCR).
A siRNA directly targeting MALAT1 (si-MALAT1; 5′-GTGAATGAGTGATAAGTAA-3′) and appropriate nontargeting siRNA (si-NC; 5′-TTCTCCGAACGTGTCACGT-3′) were bought from Geneseed Biotech Co. (Guangzhou, China). In addition, GenePharma Co. Ltd. (Shanghai, China) provided miR-205 mimics (5′-UCCUUCAUUCCACCGGAGUCUG-3′), negative control mimics (miR-NC; 5′-GGUCCGUCCGUAAUUAUCCUCC-3′), and a miR-205 inhibitor (anti-miR-205; 5′-CAGACUCCGGUGGAAUGAAGGA-3′) as well as its negative control (anti-miR-NC; 5′-CAGACUCCGGUGGAAUGAAGGA-3′). The CREB1 overexpression plasmid came from our lab. mGCs (5 × 103 cells/well) underwent transient transfection with the abovementioned constructs (100 nM siRNAs, mimics and inhibitors) using Lipofectamine 3000 (Invitrogen, CA, USA) based on provided protocols, with the efficiency of transfection being assessed at 48 h following transfection by quantitative real-time PCR (qRT-PCR).
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