Effects of microRNA-708 on Epithelial-Mesenchymal Transition, Cell Proliferation and Apoptosis in Melanoma Cells by Targeting LEF1 through the Wnt Signaling Pathway

Abstract

This study was conducted in order to elucidate the role microRNA-708 (miR-708) plays between proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) involving melanoma cells by targeting using LEF1 through the Wnt signaling pathway. Male Kunming mice were selected and subsequently divided into normal and model groups to take part in this study. Following cell line selection, the B16 cells with the highest miR-708 expression were selected and assigned into the control, blank, negative control (NC), miR-708 mimic, miR-708 inhibitor, siRNA-LEF1, and miR-708 inhibitor + siRNA-LEF1 groups. A Bioinformatics Web service and dual-luciferase reporter assay were conducted in order to determine the relationship between LEF1 and miR-708. The RT-qPCR method was performed in order to detect the miR-708 expression and mRNA expressions of LEF1, β-catenin, Wnt3a, N-cadherin, Bcl-2, Bax, Caspase3, E-cadherin, and western blotting was used in order to detect the protein expressions of these genes. MTT assay, scratch test, Transwell assay, and flow cytometry were all conducted in order to detect the cell proliferation, migration, invasion, and cycle/apoptosis, respectively. LEF1 was verified as the target gene of miR-708. In comparison with the normal group, the model group had reduced expressions of miR-708, Bax, Caspase3, and E-cadherin, while showing elevated expressions of LEF1, β-catenin, Bcl-2, Wnt3a, and N-cadherin. In comparison to the blank and control groups, the miR-708, mimic, and siRNA-LEF1 groups had elevated expressions of Bax, Caspase3, and E-cadherin, while also showing enhanced cell apoptosis. The miR-708, mimic, and siRNA-LEF1 groups also had decreased expressions of LEF1, β-catenin, Bcl-2, Wnt3a, and N-cadherin, and reduced optical density value 48 h and 72 h after transfection. Besides, these two groups showed declined cell migration and invasion, as well as lengthened G0/G1 phase (increased cell number) and shortened S phase (decreased cell number). Our findings demonstrated that an overexpressed miR-708 inhibits the proliferation, invasion, migration, and EMT, but also promotes the apoptosis of melanoma cells by targeting LEF1 through the suppression of the Wnt signaling pathway.

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