EYA4 could be an indicator signifying colorectal cancer sufferers' prognoses

the expression The mRNA level of EYA4 in diseased tissues and adjacent normal tissues of CRC patients were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The association between EYA4 expression and clinicopathological characteristics was analyzed by χ 2 test. Kaplan-Meier analysis with log rank test was performed to evaluate the effects of EYA4 expression on overall survival of CRC patients. Cox regression model was applied for prognosis analysis in CRC.


Results
The mRNA level of EYA4 was signi cantly decreased in CRC tissues compared with that in the adjacent normal tissue (P < 0.01). And its expression was affected by DUKE stage (P = 0.034), differentiation (P = 0.027) and vascular invasion (P = 0.037). Survival curve showed that patients with low expression of EYA4 had a signi cantly shorter overall survival than those with high expression (log rank test, P = 0.008). Low expression of EYA4 (HR = 1.989, 95%CI = 1.090-3.62902, P = 0.025) was an independent biomarker for poor prognosis in CRC patients.

Conclusion
EYA4 expression is decreased in CRC patients and negatively correlated with aggressive tumor progression. EYA4 may be a potential prognostic biomarker for CRC.

Background
Colorectal cancer (CRC) is one of the most common malignancies in the world, being responsible for many cancer-related deaths in adult [1]. The in ammatory bowel disease (IBD), especially chronic ulcerative colitis (UC) and Crohn's colitis, can enhance the risk of CRC [2,3]. Despite of the great improvements in surgical resections as well as the integration of chemoradiotherapy and targeted therapy, the long term survival of patients with CRC is still very poor [4,5]. Many CRC cases will develop local recurrence or distant metastasis within 5 years after operation, resulting in therapeutic failures and high mortality [6]. Until now, there is no widely accepted parameter to provide reliable information for tumor progression and prognosis in CRC [7]. For this reason, it is urgently needed to identify e cient cancer-related genetic biomarkers for improving the poor prognosis of CRC.

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The Eyes Absent 4 gene (EYA4), is a member of EYA gene family which includes four members, EYA1-EYA4, in mammals [8]. The EYA family was initially identi ed in Drosophila, and played pivotal roles in eye development [9]. Growing evidences have reported that EYA4 is implicated in the formation and development of mammalian organs, regulation of cellular behaviors, apoptosis, innate immunity, DNA damage repair, angiogenesis, as well as tumorigenesis [10]. Recently the aberrant hypermethylation of EYA4 promoter has been observed in a number of cancers, such as melanomas, lung cancer, pancreatic cancer, and hepatocellular carcinoma [11][12][13][14]. It was reported that hypermethylation mediated EYA4 silence might contribute to tumorigenesis, revealing the inhibitory roles of EYA4 in carcinogenesis [14]. The anti-tumor action of EYA4 was also studied in CRC. Sung-Jin Kim et al, demonstrated that overexpression of EYA4 could lead to inhibition of cell proliferation via inducting up-regulation of DKK1 and inhibiting the Wnt signaling pathway, indicating its capacity as a tumor suppressor gene in CRC [15].
However, the clinical signi cance of EYA4 in prognosis of CRC was rarely reported.
In this study, we mainly investigated the expression pattern of EYA4 in CRC tissues samples, and its association with clinicopathological characteristics of the patients. Additionally, we further estimated the prognostic value of EYA4 in CRC.

Patients and specimens
The study population was consisted of 155 patients pathologically diagnosed with CRC, including 71 females and 84 males. The tumor tissues and adjacent normal tissues specimens were collected from the patients through surgical resection at Chinese PLA General Hospital. None of these patients had received chemotherapy or radiation therapy prior to surgery. The specimens were immediately put into liquid nitrogen after surgery and then stored at -80℃ until RNA extraction. The detail clinical information of the patients was listed in Table 1. A 5-years follow-up was conducted for the patients. In our study, we excluded the patients who were died from unexpected events or other diseases.

Statistical analysis
Statistical analyses were performed using the software of SPSS 21.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 (GraphPad, San Diego, CA, USA). Continuous data were presented as mean ± SD. The different levels of EYA4 mRNA between CRC tissues and adjacent normal tissues were analyzed with student's t test. χ 2 test was used to assess the relationship between EYA4 expression and the clinicopathological characteristics. Kaplan-Meier analysis with log rank test was used to evaluate the effects of EYA4 expression level on overall survival of CRC patients. Cox regression analysis was conducted to evaluate the prognostic value of EYA4 and clinical parameters in CRC. The difference was considered to be statistically signi cant when P value less than 0.05.

Results
The expression of EYA4 was decreased in CRC tissues To study the expression pattern of EYA4 in CRC, qRT-PCR method was applied in this study. As shown in Fig. 1, the mRNA expression level of EYA4 was remarkably downregulated in CRC tissues compared with adjacent cancer tissues (P < 0.01).

Relationship between EYA4 expression and clinicopathological characteristics of patients with CRC
To investigate whether EYA4 was involved in the development of CRC, the patients were divided into high expression group (n = 99) and low expression group (n = 56) according to the mean expression of EYA4. The expression of EYA4 was signi cantly associated with DUKE stage (P = 0.034), differentiation (P = 0.027) and vascular invasion (P = 0.037). However, no correlation was found between EYA4 expression and age, gender, lymph node metastasis or tumor location (P > 0.05 for all) ( Table 1).

Association of EYA4 expression with prognosis of CRC patients
To evaluate the prognostic potential of EYA4 in CRC, we analyzed the association between EYA4 expression and the overall survival using Kaplan-Meier analysis with the log-rank test. The survival curves showed that patients with low expression of EYA4 had a shorter overall survival than those with high expression (Fig. 2

Discussion
CRC is a frequently diagnosed malignant tumor worldwide, with high morbidity and mortality [16]. In China, the incidence of CRC shows increasing tread, posing a great threat to human health [17]. Despite of the various available treatments for CRC, the long term outcomes of CRC patients still remain unsatisfactory, which may be attributed to high occurrence of postoperative recurrence and metastasis [18]. Tumor progression prediction represents a challenge for CRC patients in clinic, due to the lack of credible biomarkers [19]. Therefore, identi cation of effective prognostic biomarkers for CRC may be a promising approach to improve management and prognosis of patients with CRC.
The development and progression of CRC is a complex process, with the involvement of a variety of molecules and signaling pathways, such as oncogene activation, suppressor gene inactivation, and epigenetic modi cations [20]. Up to now, the molecular mechanisms underlying CRC is still unclear. In order to improve the management and prognosis of CRC, various researches were devoted to explore the genetic biomarkers for prognosis prediction in CRC. For instance, Zhu et al., found that Twist1 could enhance proliferation and chemoresistance of CRC cells which might be a potential prognostic marker and a molecular therapeutic target for the cancer [21]. Zhang et al. indicated that up-regulation of nemolike kinase (NLK) was positively correlated with recurrence and distant metastasis of CRC patients, predicting poor prognosis for patients with the disease [22]. The clinical signi cance of MTA2 expression for prognosis of CRC was investigated by Ding et al. They found the expression pro le of MTA2 exhibited high in CRC tissues specimens, and signi cantly correlated with overall survival of the patients, suggesting its possible as a prognostic biomarker for CRC [23]. These cancer-related molecules might provide reliable information for tumor progression of CRC. In the current study, we investigated the prognostic value of EYA4 for CRC.
EYA4, a member of EYA family, has recently been identi ed as a tumor suppressor gene in many kinds of cancers, including hepatocellular carcinoma, acute myeloid leukemia, oral cancer, etc [14,24,25]. In the current study, we investigated the functional roles of EYA4 in CRC. We detected the expression of EYA4 at mRNA level in CRC tumor tissues and the adjacent normal tissues. Our results showed that the level of EYA4 was signi cantly down-regulated in tissues compared with adjacent normal tissues. Then we further investigated the effects of EYA4 expression level on the development of CRC. The results indicated that EYA4 was negatively associated with DUKE stage, differentiation and vascular invasion. All the data revealed the anti-tumor action of EYA4 in CRC, and its reduced expression could contribute to malignant progression of the disease.
Given its function in tumorigenesis, EYA4 was identi ed as a biomarker for several cancers. In pancreatic ductal adenocarcinoma, down-regulation of EYA4 predicted short overall survival for the patients [26]. The expression pattern of EYA4 also held referenced signi cance for prognosis of patients with intrahepatic cholangiocarcinoma [27]. In CRC, Liu et al. reported that the serum methylation levels of EYA4 were signi cantly different between stage I CRC patients and healthy individuals that might be an useful biomarker for early detection of CRC [28]. However, the prognostic value of EYA4 in CRC was still unclear. In this study, Kaplan-Meier analysis showed that patients with low expression of EYA4 had a shorter overall survival than those with high expression. According to cox regression analysis, EYA4 might be employed as an independent prognostic factor for CRC. Low expression of EYA4 predicted poor prognosis for the patients.

Conclusions
In conclusion, EYA4 is decreased in CRC patients and its expression is signi cantly correlated with tumor differentiation, metastasis and clinical stage. EYA4 may be a potential prognostic biomarker for CRC. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Y.Y. design of the work; X.D. and S.X. the acquisition, analysis, S.L. and D.T. interpretation of data; S.H. and Y.W. the creation of new software used in the work; R.L. have drafted the work or substantively revised it. All authors read and approved the nal manuscript.  Figure 1 The relative expression level of EYA4 mRNA in CRC tissues and adjacent normal tissue. The expression level of EYA4 in CRC tissues was lower than that in the normal specimens (**: suggested P<0.01).