EEF1A Acts as A Promising Novel Bio-Marker in the Prognosis of Patients With Cholangiocarcinoma

Background: The morbidity and mortality of cholangiocarcinoma (CCA) is increasing in recent years. EEF1A2 could regulate multiple biological and pathological processes. Our study was designed to investigate the clinical signicance of eEF1A2 for prognosis evaluation in CCA. Methods: The quantitative analysis for eEF1A2 in CCA specimens was performed using quantitative real-time polymerase chain reaction (qRT-PCR). The inuences of eEF1A2 expression on disease progression and survival of CCA patients were analyzed by Chi-square test and Kaplan-Meier method, respectively. Additionally, cox regression analyses were adopted to identify the potential predictive biomarker for prognosis of CCA. Results: eEF1A2 expression showed increased tendency in CCA tissues (P<0.05). The expression prole of eEF1A2 was positively correlated with TNM stage (P=0.004), lymph node metastasis (P=0.001) and distant metastasis (P=0.001). In addition, high eEF1A2 expression predicted dismal survival among CCA cases (P<0.05). EEF1A2 might be independently correlated with prognosis of CCA (HR=2.724, 95%CI=1.303-5.964, P=0.008). Conclusions: eEF1A2 may act as a predictive biomarker for clinical outcomes of CCA.


Background
Cholangiocarcinoma (CCA) is a common malignant tumor originated from the biliary epithelial cells [1,2]. According to the anatomical sites, CCA was divided into intrahepatic cholangiocarcinoma, hilar cholangiocarcinoma and distal cholangiocarcinoma by the seventh AJCC/UICC [3]. The most pathological types of CCA are differently differentiated adenocarcinoma [4,5]. CCA is characterized by concealed pathogenesis, rapid lymphatic invasion and distant metastasis, and most cases developed to advanced stages when initial diagnosis, leading to low resection rate and dismal clinical outcomes. In recent years, the incidence of CCA exhibits increasing tendency [6,7]. Even worse, CCA patients show insensitive to radiotherapy and chemotherapy [8,9], resulting in poor prognosis. Therefore, the predictive biomarkers are in urgent need to guide the clinical treatments and predictive the clinical outcomes of CCA.
Eukaryotic elongation factor 1 alpha (eEF1A) is responsible for protein synthesis. It is a kind of guanine nucleotide-binding regulatory (G protein), and it can regulate the combine of ribosomal A site and aminoacylated tRNAs in order to promote the elongation of ribosomal polypeptide [10,11]. It is reported that eEF1A may take part in multiple cellular processes such as protein translation, protein degradation, apoptosis and cell proliferation [12,13]. eEF1A, contains two isoforms, named eEF1A1 and eEF1A2. EEF1A2 gene is located in the chromosome 20q13.3 with 12 kb in length, and eEF1A2 is the protein product of eEF1A2 gene [14]. eEF1A2 may function as a regulator for protein translation [15]. Dysregulation of eEF1A2 has been reported in several human cancers, and it may play oncogenic roles in carcinogenesis [16]. The clinical investigations con rmed that the increased expression of eEF1A2 showed close association with perineural invasion, lymph node metastasis and poor prognosis [17].
However, the predictive value of eEF1A2 for clinical outcomes of CCA still remained unclear.
In our study, the expression pattern and its in uences on clinical parameters of eEF1A2 were investigated in CCA.Moreover, the survival curves and cox analyses were adopted to measure whether eEF1A2 could act as a predictive biomarker for CCA patients.

Study subjects and sample collection
In this study, 121 eligible CCA cases were recruited from the PLA Rocket Force Characteristic Medical Center. Diagnosis of CCA was made based on pathological examinations, and none of the cases had history of anti-tumor treatments, including chemotherapy, radiotherapy, or surgery. The surgical CCA tissues and corresponding non-cancerous tissues were frozen in liquid nitrogen ,and then maintained at -80℃. The CCA patients were followed up every 3 months in the rst year and then every 6 months in the subsequent two years, and then annually. The follow-up investigation lasted for ve years. The clinicopathological features of CCA patients were listed in Table 1, including gender, age, tumor location, histological grade, TNM stage, lymph node metastasis and distance metastasis.  The study permission was obtained from the Ethics Committee of the PLA Rocket Force Characteristic Medical Center. All the participants provided the written informed consents.
Quantitative analysis for eEF1A2 RNA extraction was performed by Qiagen RNeasy kits (Qiagen, Crawley, UK), and the experimental procedures were carried out according to the instructions of manufacturer. 1 µg RNA sample with high quality was used for cDNA synthesis, and the Superscript III Reverse Transcriptase (Invitrogen) was adopted. The quantitative analysis for eEF1A2 was achieved by quantitative real-time polymerase chain reaction (qRT-PCR), and the reaction was carried out by SYBR Green PCR master mix (Applied Biosystems) on the Rotor-Gene 6000 real-time genetic analyzer (Corbett Life Science, USA). GAPDH was employed as an internal reference. The speci c ampli ed primers were as follows: eEF1A2 forward: 5'-CCATGTGTGTGGAGAGCTTCTC-3', reverse: 5'-TCTCCACGTTCTTGATGACGCC-3'; GAPDH forward: 5'-GTCTCCTCTGACTTCAACAGCG-3', reverse: 5'-ACCACCCTGTTGCTGTAGCCAA-3' [18]. The relative expression of eEF1A2 was normalized against GAPDH, and analyzed by the method of 2 −ΔΔCt .

Statistical analysis
The expression level of eEF1A2 was expressed as mean ± standard deviation (SD), and its comparison between CCA and non-cancerous specimens was achieved by student's t test. The possible relationship between eEF1A2 expression and the cliincal parameters of CCA cases was con rmed via Chi-square test.
The survival curves were plotted using Kaplan-Meier method with log-rank test for CCA cases according to their expression patters of eEF1A2. The prognosis analysis was performed by Cox regression analysis to identify the potential predictive biomarker for prognosis evaluation of CCA. P < 0.05 indicated the statistical signi cance of the analysis results.

Upregulation of eEF1A2 in CCA tissues
The quantitative analysis for eEF1A2 was performed in 121 pairs of CCA tissues and corresponding normal tissues. As displayed in Figure 1, eEF1A2 expression exhibited signi cantly increased tendency in CCA tissues compared to the non-cancerous specimens (P<0.05).
In uences of eEF1A2 expression on disease progression of CCA patients The included CCA cases were divided into high expression (n=67) and low expression (n=54) groups based on their mean eEF1A2 expression in CCA tissues. Chi-square test showed that the CCA patients with high expression of eEF1A2 were more likely to undergo advanced TNM stage (P=0.004), positive lymph node metastasis (P=0.001) and distant metastasis (P=0.001). Meanwhile, the expression level of eEF1A2 had no obvious association with age, gender, tumor location or histological grade (all P>0.05) ( Table 1).

Overall survival curves and prognosis analysis
The survival curves plotted for CCA patients according to their eEF1A2 expression showed that CCA patients with high eEF1A2 expression had worse overall survival rates than those with lower eEF1A2 expression (log-rank test P=0.003) (Figure 2).
The prognostic analysis was achieved by Cox regression model. Analysis results con rmed that eEF1A2 might be independently correlated with clinical outcomes of (P=0.008). It was shown that increased expression of eEF1A2 had adversely in uence to CCA with HR of 2.724 and 95%CI of 1.303-5.694, and it also suggested an increasing risk of dying to CCA patients with up-regulation of eEF1A2.

Discussion
CCA is originated from the biliary epithelium, making up 2-3% of all gastrointestinal tumors [19]. Epidemiological data show that the prevalence of CCA is on rise year by year around the world [20,21].
Due to the lack of obvious symptoms in early stages, many CCA cases present local in ltration and lymphatic or distal metastasis when initial diagnosis, resulting in dismal outcomes. In China, the 5-year survival rate is only 5% among the patients with advanced CCA [2,22]. The predictive biomarkers were key for early screening and treatment guidance in CCA. In current study, we explored whether eEF1A1 could be employed as a biomarker for prognosis evaluation of CCA.
EEF1A is one of the four subunits of eukaryotic elongation factor 1 (EEF1), belonging to G protein family [23,24]. EEF1A has two isomerides, eEF1A1 and eEF1A2. Under normal physiological conditions, eEF1A1 widely expresses in lung and liver, but its expression was rarely detected in cardiac muscle, skeletal muscle and brain. Moreover, eEF1A2 expression was rarely observed in neither normal lung nor normal liver tissues [25,26]. Dysregulation of eEF1A2 may lead to diseases, like tumors. The altered expression of eEF1A2 was reported to be involved in several human malignancies, such as breast cancer [12], pancreatic cancer [27], ovarian cancers [28]. In this study, we explored the expression pro le of eEF1A2 in CCA.
In our study, the expression patterns of eEF1A2 was detected in CCA specimens. The quantitative analysis showed that eEF1A2 expression level was higher in CCA tissues than that in the matched noncancerous tissues. Moreover, the expression of eEF1A2 was signi cantly related to TNM stag, lymph node metastasis and distant metastasis. And the results of Kaplan-Meier survival analysis showed that CCA patients with high eEF1A2 expression were more likely to undergo poor survival rate compared to those with low expression. In addition, Cox regression analysis suggested that the expression of eEF1A2 was an independent prognostic factor for the survival of CCA patients. Taken together, high expression of eEF1A2 predicted aggressive disease progression and poor prognosis of CCA.
In the previous studies, the predictive values of eEF1A2 for prognosis had been investigated in several cancers. Duanmin et al. have shown that the increased expression of eEF1A2 in pancreatic ductal adenocarcinoma was closely associated with nodal metastasis, perineural invasion and worse prognosis [17]. In the study of Yang et al., the over-expression of eEF1A2 was con rmed to be correlated with worse outcomes in gastric cancer patients [29]. Moreover, Kawamura et al. reported that the positive expression of eEF1A2 in non-small cell lung cancer indicated poor prognosis [30]. However, upregulated eEF1A2 expression predicted prolonged survival for the cases with ovarian cancer and breast cancer [31,32], which were not in accordance with our ndings. The disparity in prognostic signi cance of eEF1A2 implied its multiple biological functions depending on cancer types with distinct genetic background. Therefore, further investigation is necessary to determine the role of eEF1A2 in the development of CCA.

Conclusions
In conclusion, the up-regulation of eEF1A2 in CCA predict aggressive disease progression and poor clinical outcomes. eEF1A2 may be a potential predictive biomarker for prognosis of CCA.

Declarations
Ethics approval and consent to participate This study was supported by the Ethics Committee of the PLA Rocket Force Characteristic Medical Center, and the experimental procedures were carried out in accordance with the World Medical Association Declaration of Helsinki.
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 materialsThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Figure 1
The expression pro les of eEF1A2 expression in CCA tissues and paired adjacent noncancerous tissues (*, P<0.05).