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

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.

F-box and WD repeat domain-containing 7 (FBXW7) is the substrate recognition component of an evolutionarily conserved SCF (complex of SKP1, CUL1, and F-box protein)-type ubiquitin ligase complexes, which has been well characterised and shown to have important roles in regulating the stability of multiple oncoprotein substrates, including cyclin E, c-Myc, Notch, c-Jun, mammalian target of rapamycin, and MCL1 (Nakayama and Nakayama, 2006;Mao et al, 2008;Welcker and Clurman, 2008;Inuzuka et al, 2011;Wertz et al, 2011). Therefore, the altered expression of FBXW7 is recognised to be one of the major causes of carcinogenesis or cancer development. We have previously revealed that loss of FBXW7 correlated with a poor prognosis in colon cancer (Iwatsuki et al, 2010). Similarly, low expression of FBXW7 has been reported to be significantly associated with poor prognoses in glioma (Bredel et al, 2005;Hagedorn et al, 2007), gastric cancer (Yokobori et al, 2009), and breast cancer (Ibusuki et al, 2011). However, significance of downregulation of this molecule in oesophageal squamous cell carcinoma (ESCC) remains unknown.
Recent studies have revealed that microRNAs (miRNA) can act as oncogenes or tumour suppressors during the development and progression of cancers through sequence-specific binding to their mRNA targets (Ambros, 2004;Zamore and Haley, 2005;Meister, 2007). These miRNAs have an important role in a wide variety of complex biological processes, including cellular development and differentiation, but investigations have only begun to clarify their significance in carcinogenesis (Calin et al, 2004;Croce and Calin, 2005). Some researchers have noted that alterations in the miRNA expression profile strongly affect the progression of human tumours and the prognosis of the patients (Yanaihara et al, 2006;Bloomston et al, 2007;Schetter et al, 2008;Ueda et al, 2010). Although our previous study revealed that miR-21 is significantly overexpressed in ESCC (Hiyoshi et al, 2009), there have been few reports concerning the miRNA profiles in ESCC.
It was recently reported that microRNA-223 (miR-223) targets the FBXW7 mRNA 3 0 -untranslated region, and that overexpression of miR-223 significantly reduces FBXW7 mRNA levels, increases endogenous cyclin E protein and activity levels, and increases genomic instability (Xu et al, 2010). Nevertheless, to our knowledge, no study has been reported on the relationship between the expression levels of miR-223 and FBXW7 in clinical samples of solid tumours. However, no correlation between miR-223 and FBXW7 has yet been elucidated in ESCC.
In the present study, we examined the correlation between the expression levels of miR-223 and immunohistochemical staining for the FBXW7 protein in 109 consecutive ESCC samples, and we also investigated the prognostic significance of the expression of miR-223. Moreover, we identified FBXW7 as a functional downstream target of miR-223 in vitro.

Patients and tissue samples
Primary ESCC tissue samples and samples of matched normal oesophageal epithelium were obtained from 109 patients who underwent oesophageal resection without preoperative treatment in the Department of Gastroenterological Surgery, Kumamoto University Hospital from 2000 to 2007. Written informed consent was obtained from all patients. The clinicopathological characteristics, including age, gender, pathology, differentiation, and tumour-node metastasis classification were available for all patients. Survival was measured from the time of oesophageal resection and death was the endpoint. The patient prognoses were examined in March 2011. The median observation time for survival was 31 months and it ranged from 1 to 132 months. The study was approved by the medical ethics committee of Kumamoto University.

ESCC cell lines
The human ESCC cell lines TE1, TE4, TE6, TE8, TE9, TE10, TE14, and TE15 were provided by the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University, Japan. All cells were grown in RPMI 1640 (Cambrex, East Rutherford, NJ, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA), 2 mmol l À1 glutamine, 100 units of penicillin per ml, and 100 mg of streptomycin per ml (Cambrex), and were incubated at 371C in a humidified chamber supplemented with 5% CO 2 .

miRNA isolation
The miRNAs were extracted from formalin-fixed, paraffinembedded oesophageal tissues using a RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Ambion, Austin, TX, USA), according to the manufacturer's instructions. The purity and concentration of all RNA samples were evaluated by their absorbance ratio at 260/280 nm determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA).

Quantitative real-time reverse transcription-PCR (qRT -PCR)
The expression levels of miR-223 were determined by TaqMan qRT-PCR using TaqMan microRNA assay kits (Ambion, USA) according to the manufacturer's protocols, as described previously (Hiyoshi et al, 2009). The miR-223 expression was normalised to that of RNU6B, a small nuclear RNA. The expression levels of FBXW7 were determined using primers and probes that were designed using the Universal Probe Library (Roche Diagnostics, Mannheim, Germany) following the manufacturer's recommendations. The primer sequences used for real-time PCR were as follows: FBXW7 forward 5 0 -AAAGAGTTGTTAGCGGTTCTCG-3 0 , reverse 5 0 -CCACATGGATACCATCAAACTG-3 0 and universal probe #78; 18s rRNA forward 5 0 -TGGAGGAGACGTTCCAGTGT-3 0 , reverse 5 0 -GATCTGTCCAGGCAGTCCTT-3 0 and universal probe #17. All qRT -PCR reactions were run using a LightCycler 480 System II (Roche Diagnostics, USA). The relative amounts of miR-223 and FBXW7 were measured using the 2 ÀDDCT method. All qRT-PCR reactions were performed in triplicate.

Immunohistochemical analysis
The immunohistochemical studies for FBXW7, c-Myc, and c-Jun were performed on formalin-fixed, paraffin-embedded surgical sections obtained from 109 patients with ESCC. Tissue sections of 5 mm thickness were deparaffinized and pre-treated for antigen retrieval by autoclave heating in 10 mM sodium citrate buffer (pH 9.0) for 15 min. These sections were blocked for endogenous peroxidase activity with 3% H 2 O 2 in methanol for 60 min and then washed in phosphate-buffered saline. The sections were incubated in primary mouse monoclonal anti-FBXW7 (1 : 100, Abnova Corporation, Taipei, Taiwan), mouse monoclonal anti-c-Myc (sc-40, 1 : 100, Santa Cruz Biotechnology, Fremont, CA, USA), or rabbit polyclonal anti-c-Jun (sc-1694, 1 : 50, Santa Cruz Biotechnology) antibody. Tissue sections were immunohistochemically stained using ENVISION reagents (ENVISION þ Dual Link System-HRP, Dako Cytomation, Glostrup, Denmark). All sections were counterstained with haematoxylin. The staining assessment was independently carried out by two authors (JK and YB) without any information about the patients' clinicopathological characteristics or prognosis.

Transfection of miRNA
The cells were transfected with 20 nM Pre-miR miRNA Precursor Molecule pre-223 (pre-miR-223) and 100 nM anti-miR miRNA inhibitor anti-223 (anti-miR-223) (Applied Biosystems) using the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. The specificity of the transfection was verified using the Pre-miR miRNA Precursor Molecule Negative Control #1 (control pre-miR) and Anti-miR miRNA Inhibitors Negative Control #1 (control anti-miR) (Applied Biosystems). The expression levels of miR-223 and FBXW7 were quantified 72 h after transfection, and the cells were used for a western blot analysis.

Statistical analysis
All experiments were repeated at least three times. Continuous variables were expressed as the means ± s.d. The relationship between the expression of miR-223, the FBXW7 protein, and the patient clinicopathological characteristics was analysed using Student's t-test or a w 2 -analysis. The overall survival curves were plotted according to the Kaplan -Meier method, and the generalised log-rank test was applied to compare the survival curves. The findings were considered to be significant at a P-value o0.05. All statistical analyses were performed using the SPSS v. 13.0 software program (SPSS, Inc., Chicago, IL, USA).

Clinicopathological significance of miR-223 in ESCC patients
The expression levels of miR-223 were examined in 109 ESCC clinical samples using qRT -PCR, with quantified values used to calculate miR-223/U6B ratios. The mean expression levels of miR-223 in cancerous tissue specimens were significantly higher than those in non-cancerous tissues (Po0.001; Figure 1A). We divided the 109 ESCC patients into two groups according to the ratio of their cancer/normal tissue expression levels of miR-223, as X1.0 or o1.0 for the cancer/noncancerous tissues expression levels of miR-223. There were 74 cases (67.9%) in the high miR-223 group and 35 cases (32.1%) in the low miR-223 expression group. The association between the patient clinicopathological characteristics and miR-223 expression is summarised in Table 1. There were significant differences in gender (P ¼ 0.008), tumour size (P ¼ 0.042), and depth of tumour invasion (P ¼ 0.030) between the groups.
The relationship between the expression of miR-223 and immunohistochemical staining for the FBXW7 protein in ESCC tissues A fragment of the FBXW7 3 0 -untranslated region contained three putative miR-223 binding sites as determined by a computational analysis using miRNA target prediction programs such as Target Scan (http://www.targetscan.org) and miRanda (http:// www.microrna.org) (Supplementary Figure 1).
We examined the FBXW7 protein expression level by an immunohistochemical analysis in the samples from ESCC patients. To evaluate the FBXW7 expression, the complete H score was semiquantitatively calculated by summing the products of the percentage of cells stained at a given staining intensity (0 -100) and the staining intensity score (0, none; 1, weak; 2, moderate; and 3, intense). We found an inverse correlation between the expression levels of miR-223 and FBXW7 in 109 clinical samples of ESCC. High levels of miR-223 were associated with low FBXW7 expression (Pearson correlation, r ¼ À0.336; Po0.01; Figure 1B).

The prognostic significance of miR-223 and FBXW7 in ESCC
An analysis of 5-year overall survival demonstrated that the high miR-223 expression group had a significantly poorer prognosis than the low expression group (P ¼ 0.034; Figure 2A). Similarly, the negative FBXW7 group had a significantly poorer prognosis of 5-year overall survival than the positive group (P ¼ 0.023; Figure 2B). In a univariate Cox regression analysis, compared with the low miR-223 expression group, the high miR-223 expression group experienced a significantly higher overall mortality (hazard   Table 2). In the univariate analysis, other significant prognostic factors for cancer-specific survival included lymph node metastasis (P ¼ 0.008), lymphatic invasion (P ¼ 0.002), and FBXW7 expression (P ¼ 0.023). In a multivariate Cox regression analysis for overall survival, including age at operation, N status, venous invasion, and miR-223 expression, high miR-223 expression was revealed to be an independent prognostic factor (multivariate hazard ratio 2.425; 95% confidence interval, 1.205 -4.878; P ¼ 0.013; Table 2).

The clinicopathological significance of FBXW7
We used the H score to evaluate the FBXW7 expression level and defined a final staining score of 450 as positive for FBXW7. Among the 109 ESCC patients, 39 patients (35.8%) showed positive staining for FBXW7. The associations between the patient clinicopathological characteristics and FBXW7 are summarised in Supplementary Table 1. There were no significant differences in the patient clinicopathological characteristics.

The relationship between FBXW7, and c-Myc and c-Jun in ESCC tissues
We examined the association between the FBXW7 protein expression, and c-Myc and c-Jun protein expression levels in the   samples from ESCC patients. When the FBXW7 protein was expressed at high levels, the expression levels of the c-Myc and c-Jun proteins were below the limits of detection in the miR-223 low expression cases ( Figures 3A -C). In contrast, in cases with low FBXW7 protein expression, a strong expression of the c-Myc and c-Jun proteins was noted in the cases with high miR-223 expression ( Figures 3D -F).

There is an inverse correlation between miR-223 and FBXW7 in vitro
Across all the eight cell lines tested, there was a significant inverse correlation between the expression levels of miR-223 and FBXW7 mRNA (Pearson correlation, r ¼ À0.855; P ¼ 0.007; Supplementary  Figure 2). TE6 and TE15 cells were used to evaluate the effects of    the upregulation of miR-223, and TE4 and TE14 cells were used to examine how the downregulation of miR-223 affected the FBXW7 expression. FBXW7 mRNA significantly decreased when cells were transfected with pre-miR-223, compared with those transfected with the negative control ( Figure 4A). Furthermore, we found that the protein expression levels of c-Myc and c-Jun were enhanced after pre-miR-223 treatment by a western blot analysis. In contrast, the TE4 and TE14 cells transfected with anti-miR-223 showed a decrease in the miR-223 expression, compared with the negative control-treated cells. The FBXW7 mRNA level was significantly increased in the cells transfected with anti-miR-223, compared with those transfected with the negative control, and the protein expression levels of c-Myc and c-Jun were deregulated in these cells ( Figure 4B).

DISCUSSION
In our present study, we found that miR-223 was significantly overexpressed in human ESCC tissue compared with the corresponding normal tissue (Po0.001), and that the patients with a high miR-223 expression had a significantly poorer prognosis than those with a low expression (P ¼ 0.034). We also provide evidence that a negative association exists between the expression of miR-223 and the FBXW7 protein in ESCC patients (Pearson correlation, r ¼ À0.336; Po0.01), and revealed that the miR-223 expression responds to alterations in the c-Myc and c-Jun protein levels as regulated by the FBXW7 pathway in vitro. These findings suggested that the overexpression of miR-223 correlates with the poor prognosis of ESCC, possibly because of repression of the function of the FBXW7 protein.
Loss of FBXW7 function is known to be associated with the dysregulation of several cell cycle regulators, including cyclin E and c-Myc (Welcker et al, 2003). In oesophagaeal cancer, amplification and overexpression of these regulators has been thoroughly investigated, and their clinical significance has been reported. Cyclin E, a maintainer of the cell cycle restriction point, is significantly overexpressed in mucosal invasive ESCC compared with normal mucosa (Ohbu et al, 2001). The amplification of c-Myc was more frequently found in advanced stages of ESCC than in early stages (Bitzer et al, 2003). Therefore, the regulation of FBXW7 may have an important role in the carcinogenesis and progression of ESCC. In this study, there was an inverse correlation between FBXW7, and c-Myc and c-Jun in ESCC samples as indicated by an immunohistochemical analysis. Moreover, an in vitro assay demonstrated that there was a decrease in the FBXW7 expression when miR-223 was overexpressed, which gave rise to an abnormal accumulation of the c-Myc and c-Jun proteins.
miR-223 has been recently reported to have a potential role in tumourigenesis through repressing the function of FBXW7, and the overexpression of miR-223 has been shown to significantly reduce the FBXW7 mRNA levels, while increasing both the endogenous cyclin E protein and activity levels, as well as genomic instability (Xu et al, 2010). Moreover, a recent report identified miR-223 as an E2F1 transcriptional target (Pulikkan et al, 2010), and E2F1 and miR-223 comprised an autologous negative feedback loop. These facts indicate that miR-223 is one of the key players in cell cycle regulation at the G1 -S transition. In addition, miR-223 has been reported to act as an oncogene in several solid tumours, including gastric, ovarian, and bladder cancers (Gottardo et al, 2007;Laios et al, 2008;Petrocca et al, 2008). Moreover, in gastric cancer, the expression level of miR-223 was reported to be a prognostic marker (Li et al, 2010). On the other hand, there has been another report suggesting that miR-223 acts as a tumour suppressor by directly targeting Stathmin1 to stimulate the development and progression of hepatocellular carcinoma (Wong et al, 2008). In addition, Li et al (2011) revealed the oncogene Artemin to be a target of miR-223 and the overexpression of miR-223 decreased the migration and invasion of oesophageal carcinoma cells. Therefore, on the basis of their findings, miR-223 may have a tumour-suppressor function in oesophageal carcinoma. However, in the current study, on the basis of an investigation of 109 ESCC clinical samples, we showed that miR-223 was significantly overexpressed in the tumour compared with the corresponding normal tissue. We also found that the overexpression of miR-223 correlated with tumour advancement and a poor prognosis. Moreover, in a series of gain-of-function and loss-of-function investigations, we found that these effects may be due to the downregulation of the tumour-suppressor FBXW7, which was targeted by miR-223. The decrease in the expression of FBXW7 resulting from the overexpression of miR-223 gave rise to the abnormal accumulation of c-Myc and c-Jun proteins. It is well known that one miRNA can regulate many targets and, therefore, it may be possible that the same miRNA may have opposite roles in the progression of cancer in different tissues (Shenouda and Alahari, 2009). As miR-223 also targets other genes, some are oncogenes whereas others are tumoursuppressor genes, further analyses are needed to elucidate the full spectrum of miR-223 functions.
Although we suggested that miR-223 regulates FBXW7, 16 out of 74 samples with high miR-223 expression still showed FBXW7 expression and 12 out of 35 samples with low miR-223 expression did not show FBXW7 as shown in Table 1. The relationship between miR-223 and FBXW7 was therefore not completely inverse. To explain this finding, we speculate that not only miR-223 but also various other mechanisms, have effects on the expression of FBXW7, such as epigenetic transcriptional regulation (Gu et al, 2008), the loss of genetic alteration (Iwatsuki et al, 2010), the status of p53 mutation (Yokobori et al, 2009), or the regulation by other miRs (miR-25, 27a, 92a) (Xu et al, 2010).
In conclusion, the present study at first indicates that a high expression level of miR-223 had a significant adverse impact on the survival of ESCC patients through repression of the function of FBXW7.