Sirna neat1

Human normal osteoblast cell line hFOB1.19 and osteosarcoma cell lines Saos2, MG63, U2OS, SJSA1, and HOS were purchased from ATCC (Manassas VA). Cells were cultured in DMEM containing 10% FBS (Beyotime, Nantong, China), 100 μg/ml streptomycin, and 100 IU/ml penicillin (Invitrogen, USA), and maintained under 5% CO₂ at 37°C. NEAT1 overexpression plasmid, NEAT1 siRNA, miR‐34a‐5p mimics and miR‐34a‐5p inhibitor were all constructed by GenePharma (Shanghai, China). Transfection was performed using Lipofectamine 2000 (Invitrogen, CA).

Transient transfections were conducted using the Lipofectamine® 2000 Reagent (Thermo Fisher, USA) following the instructions from the kit. Cells were seeded onto six-well plates (3 × 10⁵ per well) for 24 hours. NEAT1 overexpression vector was constructed from GenePharma (Shanghai,
China). NEAT1 siRNA, miR-410-3p and their negative controls were designed and synthesized from GenePharma (Shanghai, China). The sequences used in this study were: siNEAT1: 5′- GGGACAGACAGGGAGAGATG-3′; siRNA negative control: 5′- UGGUACUGGAUCCUACCUUUCCGUA-3′. miR-410-3p mimic: 5′-AAUAUAACACAGAUGGCCUGU-3′. Plasmid DNA was transfected at 1 µg/ml. siRNA or miRNAs was transfected at concentration of 25 nM. Subsequent experiments were performed 48 hours after transfections.

Mouse liver cell line AML-12 was purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM-F12 medium containing 10% fetal bovine serum (FBS). Upon cell adherence, they were passaged the second day, and those in the logarithmic growth phase were used in the experiments.
AML-12 cells were stimulated by 100 mM ethanol for 24 h alone, or stimulated by 100 mM ethanol for 24 h, followed by transfection of siRNA-NEAT1 negative control (NC), siRNA-NEAT1, miR-129-5p mimic NC, miR-129-5p mimic, NEAT1 overexpression vector (overexpressed (oe)-NEAT1) together with miR-129-5p mimic NC, or oe-NEAT1 together with miR-129-5p mimic. The siRNA-NC, siRNA-NEAT1, mimic-NC, miR-129-5p mimic and oe-NEAT1 were all acquired from GenePharma Co., Ltd, (Shanghai, China).
AML12 cells were seeded in 12-well plates and cultured for 24 h before transfection. The transfection was conducted by Lipofectamine 2000 reagent (Invitrogen Inc., Carlsbad, CA, USA) when the cell confluence reached 70–90%. Transfected for 6 h, the medium was changed and cells were stimulated by 100 mM ethanol for 24 h. Finally, cells and cell supernatant were collected for the subsequent cell experiments.