Overexpression of microRNA-223 regulates the ubiquitin ligase FBXW7 in oesophageal squamous cell carcinoma

Abstract

Background: F-box and WD repeat domain-containing 7 (FBXW7) is a cell cycle regulatory gene whose protein product ubiquitinates positive cell cycle regulators such as c-Myc, cyclin E, and c-Jun, thereby acting as a tumour-suppressor gene. This study focused on microRNA-223 (miR-223), which is a candidate regulator of FBXW7 mRNA. The aim of this study was to clarify the clinical significance of miR-223 and FBXW7 in oesophageal squamous cell carcinoma (ESCC) patients, and to elucidate the mechanism by which FBXW7 is regulated by miR-223.

Methods: The expression levels of miR-223 and the expression of FBXW7 protein was examined using 109 resected specimens to determine the clinicopathological significance. We also investigated the role of miR-223 in the regulation of FBXW7 expression in ESCC cell lines in an in vitro analysis.

Results: We found that miR-223 expression was significantly higher in cancerous tissues than in the corresponding normal tissues. There was a significant inverse relationship between the expression levels of miR-223 and FBXW7 protein. Moreover, patients with high miR-223 expression demonstrated a significantly poorer prognosis than those with low expression. On the basis of a series of gain-of-function and loss-of-function studies in vitro, we identified FBXW7 as a functional downstream target of miR-223.

Conclusion: Our present study indicates that high expression of miR-223 had a significant adverse impact on the survival of ESCC patients through repression of the function of FBXW7.

Figure 1

The correlation between the expression of miR-223 and the FBXW7 protein in ESCC patients. (A) The expression level of miR-223 in tumour tissue specimens was significantly higher than that in non-tumour tissues (P<0.001). (B) To evaluate the expression of FBXW7, the complete H score was semiquantitatively calculated (0–300). The relationship between miR-223 and FBXW7 in 109 clinical samples of ESCC indicated an inverse correlation (Pearson correlation, r=−0.336; P<0.01).

Figure 2

Kaplan–Meier curves according to the miR-223 and FBXW7 status. (A) The overall survival curves are presented according to the expression level of miR-223 in ESCC patients. Patients with high miR-223 expression had a poorer prognosis than those with low expression (log-rank (Mantel–Cox) test; P=0.034). (B) The overall survival curves according to the FBXW7 expression level in ESCC patients. The negative FBXW7 group had a significantly poorer prognosis for 5-year overall survival than the positive group (P=0.023).

Figure 3

The relationship between the expression of miR-223, and FBXW7, c-Myc, and c-Jun proteins in ESCC patients. (A–C) In the miR-223-low expression cases, the FBXW7 protein was expressed at a high level, whereas the levels of the c-Myc and c-Jun proteins were below the limit of detection in the same tissue sections. (D–F) In contrast, in the miR-223-high expression cases, the FBXW7 protein was expressed at a low level, and there was strong expression of the c-Myc and c-Jun proteins. ( × 200 original magnification, scale bar: 50 μm).

Figure 4

miR-223 gain-of-function and loss-of-function studies in ESCC cell lines. (A) The expression of the FBXW7 mRNA was significantly suppressed by transfection of cells with pre-miR-223, as confirmed by qRT–PCR in TE6 and TE15 cells. Furthermore, the expression of the c-Myc and c-Jun proteins was enhanced by treatment with pre-miR-223 as determined by a western blotting analysis. (B) In contrast, the FBXW7 mRNA level was significantly increased by transfection of TE4 and TE14 cells with anti-miR-223, and the c-Myc and c-Jun proteins were deregulated.