Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21287, USA.
Invest Ophthalmol Vis Sci. 2013 Jan 9;54(1):295-306. doi: 10.1167/iovs.12-10209.
Controlling the spread of uveal melanoma is key to improving survival of patients with this common intraocular malignancy. The Notch ligand Jag2 has been shown to be upregulated in primary tumors that metastasize, and we therefore investigated its role in promoting invasion and clonogenic growth of uveal melanoma cells.
mRNA and protein expression of Notch pathway components were measured using qPCR and Western blot in uveal melanoma cell lines. Expression of Jag2 ligand was upregulated using Jag2-GFP-MSCV constructs or downregulated by sh-Jag2 in the uveal melanoma cell lines Mel285, Mel290, 92.1, and OMM1, and the effects on growth and invasion were assessed.
Jag2 was introduced into Mel285 and Mel290 cells, which have low baseline levels of both this ligand and Notch activity. Overall growth of the Jag2-expressing cultures increased somewhat, and a significant 3-fold increase in clonogenic growth in soft agar was also noted. Introduction of Jag2 increased motility in both wound-healing and transwell invasion assays. We also observed a significant increase in Jag2 and Hes1 mRNA in invasive OMM1 cells that had passed through a Matrigel-coated filter in the transwell assay when compared with noninvading cells. Loss-of-function studies performed in 92.1 and OMM1 lines using Jag2 shRNAs showed that downregulation of the ligand significantly suppressed cellular growth, invasion, and migration.
Our data suggest that Jag2 may play an important role in promoting Notch activity, growth, and metastasis in uveal melanoma.
控制葡萄膜黑色素瘤的扩散是提高此类常见眼内恶性肿瘤患者生存率的关键。已有研究表明，在转移的原发肿瘤中，Notch 配体 Jag2 表达上调，因此我们研究了其在促进葡萄膜黑色素瘤细胞侵袭和克隆生长中的作用。
使用 qPCR 和 Western blot 检测葡萄膜黑色素瘤细胞系中 Notch 通路成分的 mRNA 和蛋白表达。通过 Jag2-GFP-MSCV 构建物上调 Jag2 配体的表达，或通过 sh-Jag2 在 Mel285、Mel290、92.1 和 OMM1 葡萄膜黑色素瘤细胞系中下调 Jag2 的表达，并评估其对生长和侵袭的影响。
Jag2 被引入 Mel285 和 Mel290 细胞中，这两种细胞的基础 Jag2 配体和 Notch 活性均较低。表达 Jag2 的培养物的总体生长略有增加，软琼脂中的克隆形成生长也显著增加了 3 倍。Jag2 的引入增加了划痕愈合和 Transwell 侵袭实验中的迁移能力。我们还观察到，在 Transwell 实验中，穿过 Matrigel 包被的滤器的侵袭性 OMM1 细胞中 Jag2 和 Hes1 mRNA 显著增加，与非侵袭性细胞相比。在 92.1 和 OMM1 系中使用 Jag2 shRNA 进行的功能丧失研究表明，配体的下调显著抑制了细胞生长、侵袭和迁移。
我们的数据表明，Jag2 可能在促进葡萄膜黑色素瘤的 Notch 活性、生长和转移中发挥重要作用。
