Department of Dermatology, Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, Nanjing, Jiangsu Province, China.
Department of Ophtalmology, Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, China.
Biomed Pharmacother. 2018 Nov;107:1020-1029. doi: 10.1016/j.biopha.2018.08.058. Epub 2018 Aug 24.
This study is intended to identify the key gene from gene expression profile and validate its role and regulatory mechanism in melanoma.
Gene expression profile of GSE3189 from GEO database was selected among which 7 are normal skin samples, 18 are benign skin lesion samples, and 45 are melanoma samples. The present study examined the 7 normal skin samples and the 45 melanoma samples. Differentially expressed genes (DEGs) between melanoma patients and health people were performed using Morpheus online tool. The 100 most differentially expressed genes (50 upregulated genes and 50 downregulated genes) were selected as hub genes. Then, expression levels and survival analysis of hub genes were conducted via GEPIA tool to choose target gene. The expression of target gene in melanoma cell lines was examined by RT-qPCR and western blotting. The biological function of target gene on cell proliferation in melanoma was measured in vitro. The predicted target of target gene was validated by dual-luciferase reporter assay and rescue experiment. The gene expression in clinical samples were determined by RT-qPCR, immunohistochemistry (IHC) and in situ hybridization (ISH). The tumor formation study was conducted in vivo.
Targeting protein for Xklp2 (TPX2) was identified as key gene in melanoma. TPX2 could promote the proliferation of melanoma cells. The dual luciferase reporter assay confirmed that miR-330-3p targets TPX2. In rescue experiment, it was proved that miR-330-3p inhibits the proliferation of melanoma cells by negatively regulating the expression of TPX2. The results in vitro were also confirmed in vivo. miR-330-3p/TPX2 pathway expressed differently between melanoma patients and health people. These differences were statistically significant (P < 0.05).
Inhibiting TPX2 by miR-330-3p suppresses the proliferation of melanoma cell lines. miR-330-3p/TPX2 pathway could be a potential therapeutic target in melanoma.
本研究旨在从基因表达谱中鉴定关键基因，并验证其在黑色素瘤中的作用和调控机制。
从 GEO 数据库中选择基因表达谱 GSE3189，其中 7 个为正常皮肤样本，18 个为良性皮肤病变样本，45 个为黑色素瘤样本。本研究检查了 7 个正常皮肤样本和 45 个黑色素瘤样本。使用 Morpheus 在线工具对黑色素瘤患者和健康人群之间的差异表达基因（DEGs）进行分析。选择 100 个差异表达最显著的基因（50 个上调基因和 50 个下调基因）作为枢纽基因。然后，通过 GEPIA 工具进行枢纽基因的表达水平和生存分析，选择靶基因。通过 RT-qPCR 和 Western blot 检测靶基因在黑色素瘤细胞系中的表达。在体外测量靶基因对黑色素瘤细胞增殖的生物学功能。通过双荧光素酶报告基因检测和挽救实验验证靶基因的预测靶基因。通过 RT-qPCR、免疫组织化学（IHC）和原位杂交（ISH）检测临床样本中的基因表达。进行体内肿瘤形成研究。
靶向 Xklp2（TPX2）的蛋白被鉴定为黑色素瘤中的关键基因。TPX2 可促进黑色素瘤细胞的增殖。双荧光素酶报告基因检测证实 miR-330-3p 靶向 TPX2。在挽救实验中，证明 miR-330-3p 通过负调控 TPX2 的表达抑制黑色素瘤细胞的增殖。体外结果在体内也得到了证实。miR-330-3p/TPX2 通路在黑色素瘤患者和健康人群中的表达不同。这些差异具有统计学意义（P < 0.05）。
通过 miR-330-3p 抑制 TPX2 可抑制黑色素瘤细胞系的增殖。miR-330-3p/TPX2 通路可能成为黑色素瘤的潜在治疗靶点。
