Hotair sirna

HaCaT (one human keratinocyte cell line) and A431, HSC-5, SCC13, and SCL‐1 (four CSCC cell lines) were bought from Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). Then the cells were cultured in DMEM (Gibco, Grand Island, NY) containing 10% FBS (Beyotime, Nantong, China) as well as 100 μg/mL streptomycin and 100 IU/mL penicillin (Invitrogen, USA) in a cell incubator with 5% CO₂ at 37 °C. HOTAIR overexpression (HOTAIR OE) plasmids, HOTAIR siRNAs, miR-326 mimics and miR-326 inhibitors were synthesized by GenePharma (Shanghai, China). On the basis of manufacturer’s protocol, Lipofectamine 2000 (Invitrogen, CA, USA) was applied to transfect cells.

Antibodies specific for total PDPK1 were purchased from Cell Signaling Technology. miR‐214‐3p mimics and inhibitors were purchased from RiboBio Co. Ltd. miR‐214‐3p, U6 primers and HOTAIR siRNAs were provided by GenePharma. HOTAIR and GAPDH primers were obtained from Life Technologies. Lipofectamine 3000 reagent was supplied by Life Technologies. SM was purchased from Chengdu Must Bio‐technology Company, which was freshly diluted to the final concentration with culture medium before experiment. A549 and PC9 cells and BEAS‐2B cells were obtained from the Chinese Academy of Sciences Cell Bank of Type Culture Collection and authenticated for the absence of mycoplasma, genotypes and morphology by using a commercial kit provided by Guangzhou Cellcook Biotech Co. Ltd. Cells were cultured in RPMI 1640 (Life Technologies) supplemented with 10% foetal bovine serum at 37°C in a humidified atmosphere containing 5% CO2. A549‐Luc and A549‐PDPK1(+/+)‐Luc cells were provided by Guangzhou Land Technology Co and cultured in a medium containing geneticin Sulfate (Life Technologies). Cells at 70% confluence were trypsinized with 0.25% trypsin and used in all in vitro experiments.

HRECs were seeded in 6-well plates or on slides and then transfected with lncRNA HOTAIR siRNA or negative control siRNA (GenePharma, Shanghai, China) using Lipofectamine RNAiMAX transfection reagent (13778-030; Invitrogen, Carlsbad, CA) according to the manufacturers’ instructions. In addition, HRECs were transfected with siRNA Nrf2 or overexpressing-Nrf2 (oe-HOTAIR) using Lipofectamine 2000 (12566014; Invitrogen) according to the manufacturer’s instructions. The sequences of siRNA are shown in Table 1.