Mouse melanoma cells were assigned into control, blank, negative control (NC), mimics, inhibitors, siRNA-PMEL, and inhibitors + siRNA-PMEL, LiC1 (Wnt signaling pathway activator), and siRNA-PMEL+ LiCl groups. and LiC1 group. In the siRNA-PMEL+ LiCl group, PMEL expression decreased. These findings indicated that overexpression of inhibits melanoma cell EMT, proliferation, migration, invasion, and promotes apoptosis by targetting PMEL through down-regulation of the Wnt signaling pathway. on the cell proliferation, epithelialCmesenchymal transition (EMT), and apoptosis of mouse melanoma cells by targetting PMEL through Wnt signaling pathway. Materials and methods Experimental animals Forty male Kunming mice (aging 3-month-old and weighing 20 2 g; specific-pathogen-free) were acquired from the Experimental Animal Center of Southern Medical University. All LEP mice were acclimatized to laboratory conditions (1 week before the experiment): the humidity was 50C60% (22C24C), the diurnal cycle was 12 h, with free access to food and water. All experimental procedures were strictly in accordance with the management and principles of use of the local experimental animals and abide by the expression in the B16, A375, WM239, and WM451 cells. The total RNA was extracted with a TRIzol Extraction Kit (15596-018, Invitrogen, CA, U.S.A.). The ratio of were as follows: predenaturation at 95C for 3 min, followed by 35 cycles denaturation at 95C for 15 s, annealing at 60C for ADX88178 30 s,and extension at 72C for 60 s. U6 was set as an internal reference for measurement. The ADX88178 relative expression of target gene [20] was measured by the 2 2?NC), mimics (transfected with mimics), inhibitors (transfected with inhibitors), siRNA-PMEL (transfected with siRNA-PMEL), inhibitors + siRNA-PMEL (transfected with inhibitors and siRNA-PMEL), LiC1 (treated with Wnt signaling pathway activator) and siRNA-PMEL + LiCl groups.MiR-136mimic served as a type of endogenous miRNAs, which could enhance the expression function of the endogenous [22]. inhibitor is a chemically modified inhibitor special to the specific target in cells [23]. Treated cells were seeded in a six-well plate 24 h before transfection. When the cell density grew to approximately 30C50%, the cells were transfected according to the instructions of Lipofectamine 2000 (11668-019, Invitrogen, CA, U.S.A.). Melanoma cells from the LiC1 group in the logarithmic growth phase were extracted and treated with 30 mmol/l LiCl for 1 day. In other groups, 250 l serum-free Opti-MEM (51985042, Gibco, Gaitherburg, MD, U.S.A.) was applied to dilute 100 pmol blank, NC, mimics, inhibitors, inhibitors + siRNA-PMEL, and siRNA-PMEL (50 nM as the ADX88178 final concentration), and cells were mixed and incubated at room temperature for 5 min. The 250 l serum-free Opti-MEM was applied to dilute 5 l of Lipofectamine 2000 and cells were mixed ADX88178 and incubated at room temperature for 5 min. Both the aforementioned cells were mixed, incubated at room temperature for 20 min, and added into the well of a cell-culture plate. Cells were cultured at 37C with 5% CO2 for 6C8 h, and then the medium was replaced. After culturing for 24C48 h, the cells were used for further experimentation. qRT-PCR Total RNA of melanoma tissues and normal tissues was extracted with an miRNeasy Mini Kit (217004, Qiagen Company, Hilden, Germany). The primers of mRNA): and to verify if PMEL was the direct target gene of mRNA in 3-UTR binding to were detected according to the method of the Dual-Luciferase Reporter Assay Reagent Kit provided by Genecopoeia (MD, U.S.A.). GloMax 20/20 Luminometer Luciferase Reporter Assay System (Promega, Madison, WI, U.S.A.) was used for testing the activity of dual luciferase. Each experiment was repeated thrice. MTT assay After 48 h of cell transfection, cells were collected for cell count. The cells were seeded in a 96-well plate with a cell density of 3 103 to 6 103 cells in each well (0.1 ml; with six repeating wells). Experiments were conducted at.