School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China.
State Key Laboratory of Optometry, Ophthalmology and Vision Science, Wenzhou Medical University, Wenzhou, Zhejiang, China.
Invest Ophthalmol Vis Sci. 2019 Oct 1;60(13):4503-4510. doi: 10.1167/iovs.18-26477.
MicroRNA-34a (miR-34a) has been implicated in many biological processes. It is downregulated in uveal melanoma, and introduction of miR-34a inhibits the proliferation and migration of uveal melanoma cells. Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) is a novel target of miR-34a identified first in retinal pigment epithelial cells. In this study, we sought to evaluate the interaction of miR-34a and LGR4 in uveal melanoma and its downstream mechanisms.
The expression of LGR4, epithelial-mesenchymal transition (EMT)-associated factors, and matrix metalloproteinase 2 (MMP2) in uveal melanoma cells was assessed by immunoblotting and immunofluorescence analysis. MicroRNA-34a mimic molecules, LGR4 small interfering RNA (siRNA), or MMP2-specific siRNA were transiently transfected into uveal melanoma cells. In vitro scratch and Transwell assays were used to evaluate the migratory and invasive potential of the resultant uveal melanoma cells.
LGR4 is upregulated in uveal melanoma cells. Introduction of miR-34a significantly decreased the expression level of LGR4. Transfection with miR-34a or knockdown of LGR4 attenuated the aggressiveness of uveal melanoma cells. In addition, there was a decrease in the expression of mesenchymal markers N-cadherin, vimentin, and Snail following miR-34a introduction or knockdown of LGR4. Finally, MMP2 was found to be a downstream effector for miR-34a and LGR4 that regulates the migration and invasion of uveal melanoma cells.
MicroRNA-34a negatively controls LGR4, thereby inhibiting the migration and invasion of uveal melanoma cells. Ultimately, both miR-34a and LGR4 impact the aggressiveness of uveal melanoma with alterations in the markers of the EMT. MMP2 is a downstream effector that influences the metastasis seen with uveal melanoma cells.
微小 RNA-34a（miR-34a）参与了许多生物学过程。它在葡萄膜黑素瘤中下调，并且引入 miR-34a 抑制葡萄膜黑素瘤细胞的增殖和迁移。富含亮氨酸重复的 G 蛋白偶联受体 4（LGR4）是在视网膜色素上皮细胞中首次发现的 miR-34a 的新型靶标。在这项研究中，我们试图评估 miR-34a 和 LGR4 在葡萄膜黑素瘤中的相互作用及其下游机制。
通过免疫印迹和免疫荧光分析评估葡萄膜黑素瘤细胞中 LGR4、上皮-间充质转化（EMT）相关因子和基质金属蛋白酶 2（MMP2）的表达。瞬时转染 miR-34a 模拟分子、LGR4 小干扰 RNA（siRNA）或 MMP2 特异性 siRNA 进入葡萄膜黑素瘤细胞。体外划痕和 Transwell 测定用于评估由此产生的葡萄膜黑素瘤细胞的迁移和侵袭潜力。
LGR4 在葡萄膜黑素瘤细胞中上调。引入 miR-34a 可显著降低 LGR4 的表达水平。转染 miR-34a 或敲低 LGR4 可减弱葡萄膜黑素瘤细胞的侵袭性。此外，在引入 miR-34a 或敲低 LGR4 后，间充质标志物 N-钙粘蛋白、波形蛋白和 Snail 的表达减少。最后，发现 MMP2 是 miR-34a 和 LGR4 的下游效应物，调节葡萄膜黑素瘤细胞的迁移和侵袭。
miR-34a 负调控 LGR4，从而抑制葡萄膜黑素瘤细胞的迁移和侵袭。最终，miR-34a 和 LGR4 均通过 EMT 标志物的改变影响葡萄膜黑素瘤的侵袭性。MMP2 是影响葡萄膜黑素瘤细胞转移的下游效应物。
