The overexpression of eyes absent (Eya) 2 has been found in several human cancers. However, its biological roles and clinical significance in human astrocytoma have not yet been explored. This study investigated the clinical significance and biological roles of Eya2 in human astrocytoma tissues and cell lines. Using immunohistochemistry, we found Eya2 overexpression in 33 out of 90 (36.7%) astrocytoma specimens. The rate of Eya2 overexpression was higher in grade III–IV (48.1%) than in grade I+II astrocytomas (21.1%). Transfection with an Eya2 expression plasmid was performed in A172 cells with a low endogenous expression of Eya2 and the knockdown of Eya2 was carried out in U251 cells with a high endogenous expression using siRNA. Eya2 overexpres-sion induced A172 cell proliferation and invasion, while the knockdown of Eya2 using siRNA decreased the proliferation and invasion of U251 cells. In addition, we found that transfection with the Eya2 expression plasmid facilitated cell cycle progression, and that the knockdown of Eya2 inhibited cell cycle progression, accompanied by a change in the expression of cell cycle-related proteins, including cyclin D1 and cyclin E. Eya2 also positively regulated extracellular signal-regulated kinase (ERK) activity and matrix metalloproteinase (MMP)9 expression. The blockade of ERK signaling using an inhibitor abolished the effects of Eya2 on A172 cell invasion and MMP9 production. In addition, we found that there was a positive correlation between Eya2 and Six1 in the astrocytoma cell lines. Immunoprecipitation revealed that Eya2 interacted with Six1 protein in the U251 cell line, which exhibited a high expression of both proteins. Eya2 failed to upregulate MMP expression in the A172 cells in which Six1 was silenced. On the whole, our data indicate that Eya2 may serve as a potential oncoprotein in human astrocytoma. Eya2 regulates astrocytoma cell proliferation and invasion, possibly through the regulation of ERK signaling.

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