The Interplay of MicroRNA-34a, LGR4, EMT-Associated Factors, and MMP2 in Regulating Uveal Melanoma Cells.

Purpose: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. Methods: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. Results: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. Conclusions: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.

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