The human AURKA-specific small interfering RNA (siRNA) sequence is 5′-GAAAGCTCCACATCAATAA-3′, designed with an online software of Invitrogen using AURKA sequence (GeneBank code: NM_003600) as a reference. The non-silencing (NS) sequence (5′-TTCTCCGAACGTGTCACGT-3′) was used as scrambled control that has been widely used (13). The short hairpin RNA (shRNA) cassette against AURKA is 5′-CCGGCAGAAAGCTCCACATCAATAATTCAAGAGA TTATTGATGTGGAGCTTTCTGTTTTTG-3′, with two cohesive ends for ligation into the pGCSIL-GFP vector. The double stranded shRNA oligonucleotide were ligated into pGCSIL-GFP vector linearized by restriction enzyme EcoRI and AgeI.
Next, lentiviral vector that expressed the AURKA-specific siRNA or negative control siRNA, together with pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into HEK293T cells with Lipofectamine 2000 for lentivirus generation, according to the manufacturer’s instructions (Invitrogen). After 48 h of transfection, the lentiviral particles were harvested and purified with ultracentrifugation. Due to the produced lentiviruses carrying green fluorescence protein (GFP), the viral titer was determined by counting green cells with serial dilutions under fluorescence microscopy at 5 days after infection. For lentivirus infection, H1299 and A549 cells were grown in 6-well plates at 70–80% confluence and infected with AURKA-specific siRNA lentivirus or control lentivirus at MOI of 20. Five days after infection, cells expressing GFP protein were observed using fluorescence microscopy to determine the infection efficiency. There were two experimental groups for each cell line: LV-AURKA infected cells (AURKA-siRNA) and LV-NS infected cells (scr-siRNA). Full paper
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