Lentiviral vector construction and transfection

The lncRNA RANKL sequence (5'-CAGAAGATGGCACTCACTGCA-3') was generated by Genewiz, Inc. Recombination was achieved using temporary calcium phosphate-mediated transfection of 293T cells (Cell Bank of Type Culture Collection of Chinese Academy of Sciences). The lentiviral vector was subcloned using plasmids and cells were transfected with these plasmids using a Lentiviral Packaging mix (packaging vector: Envelope=1:10) (Shanghai GenePharma Co., Ltd.) according to the manufacturer's protocol. Briefly, cells were transfected with 1 µg lentiviral vector-green fluorescent protein (pLV-GFP) or pLV-RANKL (Shanghai GenePharma Co., Ltd.) at 37˚C on a 10 cm culture plate. Vectors were collected from the supernatants on days 2 or 3 post-transfection. Cells were then transferred to fresh DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and incubated at 37˚C for 24 h with various concentrations of lentivirus (10⁷, 10⁸ and 10⁹ transducing U/ml). Pure infected cells were selected for GFP using flow cytometry and the data were analyzed with the Guava EasyCyte^™ 8 software (EMD Millipore). A total of 98% of cells were reportedly positive for GFP. A549/DPP cells were transfected using the same method. Cells selected using G418 (Sigma-Aldrich; Merck KGaA) were considered to exhibit lncRNA RANKL overexpression according to the manufacturer's protocol. Empty vector was used as a negative control (NC). The current study was approved by Cangzhou Central Hospital (Cangzhou, China).