Rna lipofectamine 2000

LNCaP cells were seeded at a density of 2.0 × 10⁵ cells/well in a 6- or 96-well culture plate (COSTAR#3516; Corning, Inc., Corning, NY) and transfected when cell confluency reached 70%. RNA Lipofectamine 2000 (Invitrogen) was used to transfect the cells with small interfering RNAs (siRNAs) to SENP1 or CDX2 according to the manufacturer's instructions. Discarded the transfection after 6 h.Then, the cells were washed with serum-free DMEM, and cultured in DMEM supplemented with 10% FBS. At 48 h after transfection, the cells were harvested for further studies. The siRNAs were designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). SiRNA for SENP1 sense: 5′-GAAACAGCCGAAGCCUUUAdTdT-3′; anti-sense: 5′-UAAAGACUUCGGCUGUUUCdTdT-3′; and siRNA for CDX2: sense 5′-GAAGAAGTTGCAGCAGCAA-3′; anti-sense: 5′-UUGCUGCUGCAACUUCUUC-3′.

The experimental culture groups included 1) untransfected LNCaP and RWPE-1 cells (control groups), 2) cells transfected with pmiR-145 or miR-145 mimics (1.6 μg/ml and 50 nM, respectively), 3) cells transfected with the scrambled nucleotide sequence and empty vector (negative control or NC groups, 50 nM), 4) cells transfected with a miRNA inhibitor (NI group, 100 nM), 5) a negative control for NI (NCI group, 50 nM), 6) cells transfected with siRNA PCGEM1 sequence (siRNA PCGEM1 group, 50 nM). Cells in log phase growth were seeded on 6-well culture plates (2 × 10⁵ cells/well) and transfected when the cell fusion rate reached 70%. The DNA Lipofectamine 2000 or RNA Lipofectamine 2000 compound was added according to the manufacturer’s instructions (Invitrogen). After 6 h, the transfection medium was discarded. Cells were washed with serum-free RPMI 1640 and then cultured in RPMI 1640 supplemented with 10% FBS.