Zehavi Liron, Schayek Hagit, Jacob-Hirsch Jasmine, Sidi Yechezkel, Leibowitz-Amit Raya, Avni Dror
Center for Cancer Research Sheba Medical Center, Tel Hashomer, Israel.
Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
Mol Cancer. 2015 Mar 26;14:68. doi: 10.1186/s12943-015-0338-9.
The incidence of cutaneous malignant melanoma continues to rise, and once the disease metastasizes it is almost inevitably fatal. We recently reported that a large miRNAs cluster on human chromosome 14q32, implicated in many types of cancers, is significantly down-regulated in melanoma. miR-377, one of the miRNAs located within this cluster, was studied here.
qRT-pCR was used to quantify miR-377 levels in melanoma cell lines and samples. Melanoma cell lines ectopically expressing miR-377 were generated by stable transfection, mRNA expression was assessed using mRNA arrays and protein expression was assessed by Western blot analysis. Potential targets of miR-377 were identified through luciferase reporter assays. Cellular proliferation, migration and soft-agar colony formation were monitored in control and miR-377-expressing cells using cell biology techniques.
miR-377 is expressed in normal melanocytes but not in melanoma cell lines or samples. Its ectopic stable expression in melanoma cell lines decreased their proliferative and migratory capacity and their colony-forming capability. mRNA arrays of melanoma cells over-expressing miR-377 pointed to several down-regulated mRNAs that have putative binding sites for miR-377 in their 3'UTR, of which both E2F3 and MAP3K7 were found to be direct targets of miR-377. E2F3, a potent transcriptional inducer of cell-cycle progression, was found to be elevated in melanoma cell lines, but decreased following ectopic expression of miR-377. Ectopic miR-377 also led to a decrease in the activity of a reporter plasmid containing three E2F DNA-binding sites linked to a luciferase cDNA sequence, demonstrating that miR-377 down-regulates E2F3-induced transcription. MAP3K7 (known as TAK1), a serine/threonine kinase along the MAPK signaling pathway, was over-expressed in melanoma but decreased following ectopic expression of miR-377. MAP3K7 is involved in the activation of NF-κB. MiR-377 over-expression led to decreased activity of a reporter plasmid containing two NF-κB DNA-binding sites and to decreased output along the NF-kB signaling pathway.
Our results suggest that miR-377 is an important negative regulator of E2F and MAP3K7/NF-kB signaling pathway in melanoma cells; it is tempting to speculate that its silencing in melanoma promotes the tumorigenic and metastatic potential of the cells through activation of these pathways.
皮肤恶性黑色素瘤的发病率持续上升，一旦发生转移几乎必然致命。我们最近报道，人类染色体14q32上一个与多种癌症相关的大型miRNA簇在黑色素瘤中显著下调。本研究对该簇内的miRNA之一miR - 377进行了研究。
采用qRT - pCR定量黑色素瘤细胞系和样本中miR - 377的水平。通过稳定转染构建异位表达miR - 377的黑色素瘤细胞系，利用mRNA芯片评估mRNA表达，通过蛋白质印迹分析评估蛋白质表达。通过荧光素酶报告基因检测鉴定miR - 377的潜在靶标。使用细胞生物学技术监测对照细胞和表达miR - 377的细胞的细胞增殖、迁移及软琼脂集落形成情况。
miR - 377在正常黑素细胞中表达，但在黑色素瘤细胞系或样本中不表达。其在黑色素瘤细胞系中的异位稳定表达降低了细胞的增殖和迁移能力以及集落形成能力。对过表达miR - 377的黑色素瘤细胞进行mRNA芯片分析，发现多个下调的mRNA，其3'UTR中有miR - 377的假定结合位点，其中E2F3和MAP3K7均被发现是miR - 377的直接靶标。E2F3是细胞周期进程的强效转录诱导因子，在黑色素瘤细胞系中表达升高，但在miR - 377异位表达后降低。异位表达miR - 377还导致含有三个与荧光素酶cDNA序列相连的E2F DNA结合位点的报告质粒活性降低，表明miR - 377下调E2F3诱导的转录。MAP3K7（又称TAK1）是丝裂原活化蛋白激酶信号通路中的一种丝氨酸/苏氨酸激酶，在黑色素瘤中过表达，但在miR - 377异位表达后降低。MAP3K7参与NF - κB的激活。miR - 377过表达导致含有两个NF - κB DNA结合位点的报告质粒活性降低，并导致NF - κB信号通路的输出减少。
我们的结果表明，miR - 377是黑色素瘤细胞中E2F和MAP3K7/NF - κB信号通路的重要负调控因子；推测其在黑色素瘤中的沉默通过激活这些通路促进了细胞的致瘤和转移潜能。
