Department of Pediatric Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China.
Department of Dermatology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China.
J Cell Physiol. 2020 Mar;235(3):2161-2170. doi: 10.1002/jcp.29122. Epub 2019 Aug 6.
MicroRNAs (miRNAs) have been validated as critical regulators in the development of melanoma. miR-140 was abnormally downregulated in uveal melanoma samples. However, the expression level and roles of miR-140-5p remain unclear in melanoma for now. We speculate that miR-140-5p is abnormally expressed and may play an important role in melanoma. The expressions of miR-140-5p and SOX4 messenger RNA were determined by quantitative real-time polymerase chain reaction assays. Western blot assays were employed to detect the expression levels of SOX4, Ki67, MMP-2, MMP-7, p-β-catenin, c-Myc, cyclin D1, p65, and IκBα. Luciferase reporter assays were employed to elucidate the interaction between SOX4 and miR-140-5p. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) and transwell invasion assays were applied to evaluate capabilities of cell proliferation and invasion, respectively. Xenograft models of melanoma were established to verify the role and molecular basis of miR-140-5p. Immunohistochemical (IHC) assays were employed to measure the Ki67 and SOX4 at the protein level in xenografted melanoma tissues. Herein, these observations showed that, miR-140-5p was abnormally downregulated in melanoma tissues and cells, while SOX4 was upregulated. miR-140-5p directly targeted SOX4 and inhibited its expression in melanoma cells. miR-140-5p overexpression repressed melanoma cell proliferation and invasion and its effects were partially restored SOX4 overexpression. Moreover, miR-140-5p hindered melanoma growth in vivo by downregulating SOX4. Mechanistically, miR-140-5p suppressed activation of the Wnt/β-catenin and NF-κB pathways by targeting SOX4. Our study concluded that miR-140-5p hindered cell proliferation, invasion, and tumorigenesis by targeting SOX4 via inactivation of the Wnt/β-catenin and NF-κB signaling pathways in malignant melanoma, which provides an underlying molecular mechanism for the treatment for melanoma with miRNAs.
微小 RNA（miRNAs）已被证实为黑色素瘤发展的关键调控因子。miR-140 在葡萄膜黑色素瘤样本中异常下调。然而，miR-140-5p 在黑色素瘤中的表达水平和作用尚不清楚。我们推测 miR-140-5p 表达异常，可能在黑色素瘤中发挥重要作用。通过实时定量聚合酶链反应（qRT-PCR）检测 miR-140-5p 和 SOX4 信使 RNA 的表达。采用 Western blot 检测 SOX4、Ki67、MMP-2、MMP-7、p-β-catenin、c-Myc、cyclin D1、p65 和 IκBα 的表达水平。采用荧光素酶报告基因检测 SOX4 与 miR-140-5p 之间的相互作用。通过 MTT（3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-四唑溴盐）和 Transwell 侵袭实验分别评估细胞增殖和侵袭能力。建立黑色素瘤异种移植模型以验证 miR-140-5p 的作用和分子基础。采用免疫组织化学（IHC）检测异种移植黑色素瘤组织中 Ki67 和 SOX4 的蛋白水平。研究表明，miR-140-5p 在黑色素瘤组织和细胞中异常下调，而 SOX4 上调。miR-140-5p 直接靶向 SOX4 并抑制黑色素瘤细胞中的表达。miR-140-5p 过表达抑制黑色素瘤细胞增殖和侵袭，其作用部分通过 SOX4 过表达恢复。此外，miR-140-5p 通过下调 SOX4 抑制体内黑色素瘤生长。机制上，miR-140-5p 通过靶向 SOX4 抑制 Wnt/β-catenin 和 NF-κB 通路的激活。我们的研究表明，miR-140-5p 通过靶向 SOX4 抑制 Wnt/β-catenin 和 NF-κB 信号通路的激活，抑制黑色素瘤细胞的增殖、侵袭和肿瘤发生，为 miRNA 治疗黑色素瘤提供了潜在的分子机制。
