Introduction

MicroRNA-224(miR-224) has been reported to promote tumor growth and invasion in several cancers and the expression of miR-224 was significantly higher in clear cell RCC (CCRCC) compared with normal kidney. However, the function of miR-224 in CCRCC remains unclear. In this study, we focused on the role of miR-224 in CCRCC as well as examination of miR-224 expression pattern in tissues and identified candidate tumor suppression gene, eyes absent 4(EYA4) as target gene of miR-224.

Methods

We investigated the expression level of miR-224 in 118 CCRCC patients who underwent surgical treatment at our institution and analyzed the relationship between the expression of miR-224 and clinic-pathological parameters. Then we did functional analysis of miR-224 using RCC cells and normal cell (RPTEC). EYA4 was identified as target gene of miRNA-224 using computer algorithm. The direct interaction analysis between miR-224 and EYA4 was also performed. We investigated functional analysis of EYA4.

Results

The expression level of miR-224 was higher in CCRCC tissues compared to matched normal kidney tissues. We divided 118 patients to two groups. A high miR-224 expression was associated with worse prognosis in CCRCC patients (Fig 1). Higher miR-224 expression was only risk factor related to progression, cancer-specific death, and overall survival. After knocking down of miR-224 in RCC cells, cell viability, invasion, migration ability were significantly decreased, whereas significantly increase of cell apoptosis in cell lines. There was an inverse correlation between miR-224 and EYA4 protein expression in CCRCC patients. After overexpression of EYA4, the cell viability and invasion were significantly inhibited in EYA4-transfected cells. Percentages of apoptotic cells were significantly increased in EYA4 transfected RCC cells compared with empty vector.

Conclusions

This is the first report to demonstrate that miR-224 expression is significantly increased in CCRCC as an oncogenic function by inhibiting EYA4 expression and may be potentially useful for a prognostic biomarker. These results also suggest that miR-224 may have therapeutic potential target for the treatment of CCRCC.

Funding

none