Invitrogen lipofectamine 2000 reagent

For transient transfection, 0.5×10⁵ DU145 cells were plated in 24 well plates, 12 h prior to transfection. The pGCMV/EGFP/Neo vector (catalog no., C05001) with overexpression of miR-30c was purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The blank vector was used as negative control. DU145 human prostate carcinoma cells were transiently transfected with mature miR-30c or control (miR-NC) using Invitrogen™ Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Following the transfection of cells, BCL9 and several Wnt pathway downstream targets were detected by western blot analysis.

All cell lines used in this study were purchased from the Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, P.R. China). The cell lines included the DU-145, LNCap, and PC-3 human prostate cancer cell lines, as well as the RWPE-1 normal prostate epithelium cell line. All cell lines were cultured in RPMI-1640 medium (Takara Biotechnology Co., Ltd., Dalian, P.R. China) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Bovine pituitary extract (0.05 mg/ml; Thermo Fisher Scientific, Inc.) and human recombinant epidermal growth factor (5 ng/ml; Thermo Fisher Scientific, Inc.) were added to the culture medium of the RWPE-1 cells. The cell culture environment was thermostatic at 37°C with constant humidity and 5% CO₂. The cells were mainly seeded into six-well plates at a density of 4 × 10⁵ cells; a lower density was used depending on certain experiments. Once the cells reached 60–70% confluency, all transfections were performed with Invitrogen Lipofectamine® 2000 Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions, with the synthesized RNA mimic or negative control (NC). Transfected cells were cultured for 48 or 72 h at the same conditions described above.