Abstract: 【Abstract】 Objective To explore whether microRNA （miRNA）-181b-5p inhibits the proliferation and invasion of cutaneous melanoma cells by targeting pleckstrin （PLEK）. Methods Bioinformatics methods were used to analyze cutaneous melanoma-associated core genes; dual-luciferase reporter assay was performed to verify the targeted interaction between miRNA-181b-5p and PLEK. Oligo RNA and small interfering RNA （siRNA） were used to regulate the expression of miRNA-181b-5p and PLEK in A375 cells respectively in this experiment, and A375 cells were divided into the following groups in detail: mimic negative control group, miRNA-181b-5p mimic group, inhibitor negative control group, miRNA-181b-5p inhibitor group, PLEK siRNA group, siRNA negative control group, miRNA-181b-5p inhibitor + control siRNA co-transfection group and miRNA-181b-5p inhibitor + PLEK siRNA3 co-transfection group. After 48-hour treatment, qPCR was performed to determine the mRNA expression of miRNA-181b-5p and PLEK in A375 cells, Western blot analysis to determine the PLEK protein expression, and Transwell assay to assess the invasive ability of A375 cells; after additional 24-96 hours of culture, cell counting kit-8 (CCK8) assay was conducted to assess the proliferative ability of A375 cells. Results PLEK was the core gene for cutaneous melanoma. PLEK expression in the cutaneous melanoma in situ tissues was significantly higher than that in the paracancerous tissues （P = 0.031）, but lower than that in the metastatic tissues （P = 0.001）. Compared with human epidermal melanocytes HEMa-LP, the mRNA and protein expression of PLEK significantly increased in A375 cells （mRNA: 3.884 ± 0.156 vs. 0.997 ± 0.010, t = 18.48, P < 0.001; protein: 2.840 ± 0.301 vs. 1.029 ± 0.094, t = 5.47, P = 0.005）, but the miRNA-181b-5p expression significantly decreased in A375 cells （0.333 ± 0.042 vs. 0.967 ± 0.069, t = 7.83, P = 0.001）. Dual-luciferase reporter assay showed targeted binding of miRNA-181b-5p to PLEK. Compared with the mimic negative control group, the miRNA-181b-5p mimic group showed significantly decreased survival rate of A375 cells （48 hours: t = 7.96, P = 0.015; 72 hours: t = 7.50, P = 0.002; 96 hours: t = 7.96, P = 0.001）, and significantly decreased invasive ability of A375 cells（t = 5.07, P = 0.007）; on the contrary, the survival rate and invasive ability of A375 cells were significantly higher in the miRNA-181b-5p inhibitor group than in the inhibitor negative control group （survival rate: 24 hours, t =5.38, P = 0.013; 48 hours, t = 5.36, P = 0.013; 72 hours, t =7.63, P = 0.005; 96 hours, t = 5.99, P = 0.004; invasive ability: t = 7.24, P = 0.002）; compared with the siRNA negative control group, the proliferative and invasive ability of A375 cells significantly decreased in the PLEK siRNA group （proliferative ability: 48, 72, 96 hours, P = 0.015, 0.011, 0.001, respectively; invasive ability: t = 4.93, P = 0.008）; compared with the miRNA-181b-5p inhibitor + control siRNA co-transfection group, the miRNA-181b-5p inhibitor + PLEK siRNA co-transfection group showed significantly decreased proliferation rate and invasive ability of A375 cells （proliferation rate: 24, 48, 72, 96 hours, P = 0.042, 0.042, 0.037, 0.017, respectively; invasive ability: t = 8.52, P = 0.001）. Conclusion miRNA-181b-5p can inhibit the proliferation and invasion of cutaneous melanoma A375 cells, likely by down-regulating the PLEK expression.

Key words: Melanoma, experimental, MicroRNAs, Cell proliferation, Neoplasm invasiveness, miR-181b-5p, Pleckstrin