关键词: 黑色素瘤, 细胞迁移分析, 上皮-间质转化, 模型, 动物, A375细胞, LncRNA 068, 炎症因子

Abstract: 【Abstract】 Objective To investigate the effect of long non-coding RNA 068 （lncRNA 068） on the migration of a melanoma cell line A375, and to explore its mechanism of action. Methods From December 2015 to November 2020, 21 patients with pathologically confirmed cutaneous melanoma were collected from Department of Dermatology, Affiliated Hospital of Nantong University, and quantitative PCR （qPCR） was performed to determine the expression of lncRNA 068 in melanoma and paracancerous tissues. LncRNA 068 was overexpressed or knocked down via lentiviral transfection in A375 human melanoma cells in the following experiments. Specifically, A375 cells were divided into lentiviral vector （LV）-green fluorescent protein （GFP） group and LV-lncRNA 068 group to be transfected with a GFP-expressing LV and a LV containing lncRNA 068 respectively in the overexpression experiment, and were divided into LV-LacZ short hairpin RNA （shRNA） group and LV-lncRNA 068 shRNA group to be transfected with a LV containing the reporter gene LacZ-specific shRNA and a LV containing the lncRNA 068-targeting shRNA respectively in the low-expression experiment, with the LV-GFP group and LV-LacZ shRNA group serving as the control groups. Transwell and scratch assays were performed to evaluate cell migration, EdU cell proliferation assay and cell counting kit-8 （CCK8） assay to determine the proportion of proliferative cells and cell viability respectively, and immunofluorescence staining was conducted to evaluate epithelial-mesenchymal transformation in the above groups. Lentivirus-transfected A375 cells from the above groups were inoculated into the axillae of BALB/c nude mice, and tumor volume was measured and calculated every 3 days. After 30 days, all mice were sacrificed, and tumor tissues were resected to measure the tumor volume and weight. Cultured B16F10 cells were subcutaneously inoculated into the back and foot of BALB/c nude mice to construct mouse models of subcutaneously transplanted B16F10 melanoma. After 2 weeks, the mice were sacrificed, and qPCR and Western blot analysis were performed to determine the mRNA expression of inflammatory factors in transplanted B16F10 melanoma and paracancerous tissues, and expression of IκB kinase （IKK）/P65 signaling pathway-related proteins, respectively. Comparisons between 2 groups were done by t test, and comparisons of tumor volume and weight at different time points among groups were done by repeated measures analysis of variance. Results qPCR showed that the relative expression of lncRNA 068 was significantly lower in human melanoma tissues and transplanted B16F10 murine melanoma tissues （0.414 ± 0.109, 0.717 ± 0.041, respectively） than in the corresponding paracancerous tissues （1.050 ± 0.103, 1.011 ± 0.023, t = 19.48, 10.83, respectively, both P < 0.001）. Transwell and scratch assays both showed that the cellular migratory ability was significantly lower in the LV-lncRNA 068 group than in the LV-GFP group （both P < 0.01）, and significantly higher in the LV-lncRNA 068 shRNA group than in the LV-LacZ shRNA group （both P < 0.05）. Immunofluorescence assay showed significantly increased fluorescence intensity of E-cadherin and decreased fluorescence intensity of N-cadherin in the LV-lncRNA 068 group compared with the LV-GFP group （both P < 0.001）, but significantly decreased fluorescence intensity of E-cadherin and increased fluorescence intensity of N-cadherin in the LV-lncRNA 068 shRNA group compared with the LV-LacZ shRNA group （both P < 0.05）. In vivo tumor formation experiment in nude mice showed that there were no significant differences in the volume or weight of melanoma between the LV-lncRNA 068 group and LV-GFP group （both P > 0.05）, as well as between the LV-lncRNA 068 shRNA group and LV-LacZ shRNA group （both P > 0.05）. As qPCR and Western blot analysis showed, the mRNA and protein expression of interleukin-10 （IL-10） and claudin-1 in A375 cells were significantly higher in the LV-lncRNA 068 group than in the LV-GFP group （both P < 0.05）, but significantly lower in the LV-lncRNA 068 shRNA group than in the LV-LacZ shRNA group（both P < 0.05）. Compared with the paracancerous tissues, B16F10 melanoma tissues showed significantly decreased mRNA expression of IL-10 （P < 0.01）, but significantly increased mRNA expression of IL-6 and tumor necrosis factor-α, as well as protein expression of phosphorylated P65 and phosphorylated IKK （P < 0.01）. Conclusion Overexpression of lncRNA 068 can inhibit the migration of A375 melanoma cells, and may affect the development of inflammation and inhibit the epithelial-mesenchymal transformation by inhibiting the IKK/P65 signaling pathway.

Key words: Melanoma, Cell migration assays, Epithelial-mesenchymal transition, Models, animal, A375 cells, LncRNA 068, Inflammatory factors