Stark Mitchell S, Bonazzi Vanessa F, Boyle Glen M, Palmer Jane M, Symmons Judith, Lanagan Catherine M, Schmidt Christopher W, Herington Adrian C, Ballotti Robert, Pollock Pamela M, Hayward Nicholas K
QIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD, Australia.
School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia.
Oncotarget. 2015 Jul 10;6(19):17753-63. doi: 10.18632/oncotarget.3924.
To identify 'melanoma-specific' microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA 'pull-down' experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E-targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.
为了鉴定“黑色素瘤特异性”微小RNA（miRNA），我们采用了一种无偏倚的miRNA谱分析方法，全面研究皮肤黑色素瘤与其他实体恶性肿瘤的关系，结果发现了233种差异表达（≥2倍，p<0.05）的miRNA。在最显著差异的前20种miRNA中，有hsa-miR-514a-3p。miR-514a是参与启动黑素细胞转化和促进黑色素瘤生长的miRNA簇（miR-506-514）的成员。我们发现miR-514a在55个黑色素瘤细胞系中的38个（69%）中表达，但在34个其他实体癌中仅在1个（3%）中表达。为了鉴定miR-514a调控的靶标，我们进行了一项miR-514a-mRNA“下拉”实验，结果发现了数百个基因，包括：CTNNB1、CDK2、MC1R和NF1，这些基因以前与黑色素瘤有关。由于NF1最近与对BRAFV600E靶向治疗的获得性耐药有关，因此选择它进行功能验证。荧光素酶报告基因检测证实NF1是miR-514a的直接靶标，并且在黑色素瘤细胞系中过表达miR-514a可抑制NF1表达，这与用PLX4032处理的BRAFV600E细胞的存活率增加相关。这些数据为MAPK途径的失调提供了另一种机制，这可能导致与当前RAF靶向治疗相关的深度耐药。
