Per1 has the capacity to serve as a diagnostic biomarker for cholangiocarcinoma patient

Serum levels of Per1 in CCA patients and healthy individuals were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Chi-square test was used to evaluate the relationship between Per1 expression and clinical characteristics of patients. The diagnostic value of Per1 in CCA was estimated by establishing a receiver operating characteristic (ROC) curve.


Abstract
Background Period 1 (Per1) had been reported to be involved in the tumorigenesis and progression of human cancers. However, the clinical signi cance of Per1 in cholangiocarcinoma (CCA) was unclear. The purpose of this study was to explore the diagnostic value of serum Per1 in CCA patients.

Methods
Serum levels of Per1 in CCA patients and healthy individuals were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Chi-square test was used to evaluate the relationship between Per1 expression and clinical characteristics of patients. The diagnostic value of Per1 in CCA was estimated by establishing a receiver operating characteristic (ROC) curve.

Results
Serum Per1 level was signi cantly down-regulated in CCA patients compared to that in healthy controls (P < 0.001). Moreover, the decreased expression of Per1 was closely associated with poor histological differentiation (P = 0.040), advanced TNM stage (P = 0.035) and positive lymph node metastasis (P = 0.007). ROC curve indicated that the area under the curve (AUC) was 0.863 with a sensitivity of 88.1% and a speci city of 72.1%, revealing the high diagnostic value of serum Per1 in CCA.

Conclusions
Per1 is down-regulated in CCA and negatively correlated with tumor progression. Serum Per1 may be a potential biomarker for early screening of CCA. Background Cholangiocarcinoma (CCA) is a rare malignant tumor originating from the epithelial cells of bile duct [1] The morbidity and death rates of CCA exhibit increasing trend in the world [2]. CCA is characterized by continually in ltrative growth, high metastasis, poor response to radiotherapy and chemotherapy [3]. Until now, radical surgery remains the only effective strategy for CCA patients. However, due to the silent growth of the cancer and lack of tools for early detection, most CCA patients are diagnosed at late stages, leading to limited therapeutic effects and poor clinical outcomes [4,5]. Currently, several serum biomarkers are applied for early detection and monitoring of CCA, including CEA, CA19-9, ALP, MUC5AC, and CA-S121. However, their clinical signi cance is unsatisfactory [6]. Therefore, it is an urgent need to identify novel biomarkers for non-invasive diagnosis of CCA patients.
The circadian rhythm is a basic character of life that can regulate various physiological activities, including cell proliferation and metabolism [7]. In living organisms, circadian system is regulated by many circadian clock genes [8]. To data, several clock genes are con rmed to be implicated with circadian rhythm, such as period 1 (Per1), period 2 (Per2), period 3 (Per3), circadian locomotor output cycles kaput (Clock), etc [9]. Per1 gene is located on human chromosome 17p13.1, belonging to the Per subfamily. Growing evidences have demonstrated that Per subfamily not only plays a crucial role in the modulation of circadian rhythms, but also participates in the development of tumors [10]. The aberrant expression of Per1 was observed in several tumors, such as colorectal cancer, head and neck squamous cell carcinoma, oral cancer, etc [11][12][13]. In CCA, it was reported that Per1 was down-regulated and its over-expression could suppress cell proliferation, cell cycle progression, and induce cell apoptosis [14]. However, little was known about the diagnostic values of serum Per1 in CCA.
In this study, we sought to detect the serum levels of Per1 in CCA patients and healthy controls. Chisquare test was applied to evaluate the association of Per1 expression with clinical characteristics of patients. Then the ROC curve was plotted to estimate the diagnostic value of Per1 in CCA patients.

Study subjects and samples
This study was approved by the Ethical Committee of the hospital and the written informed consent was obtained from each participator in advance. In our study, 122 patients who were pathologically diagnosed with CCA at the PLA Rocket Force Characteristic Medical Center were enrolled. None of patients had received any treatments before blood collection. The histopathological diagnoses were con rmed by the experienced pathologists. The detailed clinicopathologic characteristics of the patients were shown in Table 1. Besides, 84 healthy individuals who were matched the cases in age and gender were recruited as healthy controls. After fasting for one night, 5 mL blood samples were collected from CCA patients and healthy controls, and centrifuged at 3000 rpm for 10 min. Then the supernate was immediately stored at -80℃ until for RNA extraction.

Statistical analysis
All statistical analyses were performed with SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5.0 software (GraphPad, San Diego, CA, USA). The data were expressed as mean ± standard deviation (SD). Student's t-test was used to evaluate the difference of Per1 expression between case and control groups. The relationship between Per1 expression and clinical characteristics of patients was analyzed via chi-square test. A receiver operating characteristic (ROC) curve was plotted to estimate the diagnostic value of Per1 in CCA patients. If P was less than 0.05, the difference was considered statistically signi cant.

Results
Demographic data of the study subjects A total of 50 female and 72 male patients with the mean age of 60.39 ± 13.45 years (range, 46-74 years) were recruited in this study. Among these CCA patients, 63 patients had well/moderate histological differentiation and 59 patients had poor differentiation. 65 cases were diagnosed at stage -and 57 cases were at stage -. There were 44 patients presenting lymph node metastasis and 60 cases with T1-T2 depth of invasion. The detailed clinical characteristics of patients were shown in Table 1.

Serum Per1 level was down-regulated in CCA patients
The relative mRNA levels of Per1 in CCA patients and healthy controls were detected by qRT-PCR. As shown in Fig. 1, serum Per1 level in CCA patients was signi cantly lower than that in healthy controls (P < 0.001).

Relationship between Per1 expression and clinical features of CCA patients
According to the mean Per1 expression, the CCA patients were divided into high expression group (n = 53) and low expression group (n = 69). Then chi-square test was applied to evaluate the relationship between Per1 expression and clinical characteristics of patients. The result showed that decreased expression of Per1 was closely associated with poor histological differentiation (P = 0.040), advanced TNM stage (P = 0.035) and positive lymph node metastasis (P = 0.007) ( Table 1). However, there was no obvious correlation between Per1 expression and age, gender or depth of invasion (P > 0.05 for all, Table 1).

The diagnostic value of Per1 in CCA patients
To assess the diagnostic value of Per1 in CCA, a ROC curve was established. It indicated that serum Per1 could be a forceful biomarker for discriminating CCA patients from healthy controls. The area under the curve (AUC) was 0.863 (95%CI = 0.816-0.910, P < 0.001) with a sensitivity of 88.1% and a speci city of 72.1% (Fig. 2). The cutoff value of Per1 mRNA for CCA detection was 5.02.

Discussion
Early diagnosis remains a great challenge for CCA patients in clinic. In the present study, we investigated the diagnostic value of serum Per 1 in CCA. The relative mRNA level of serum Per1 in CCA patients was signi cantly lower than that in healthy controls. Furthermore, patients with decreased expression of Per1 were more easily to undergo poor histological differentiation, advanced TNM stage and positive lymph node metastasis. The result of ROC curve analysis indicated that serum Per1 might be a promising biomarker for the diagnosis of CCA with high sensitivity and speci city.
The Per genes are a subgroup of core clock genes which are involved in various biological processes via regulating circadian rhythm [15]. In human, there are three identi ed Per family members including, Per1, Per2 and Per3. Accumulating evidences have demonstrated that the Per family members were involved in carcinogenesis and development of malignancies. For instances, in lung cancer, down-regulation of Per2 might lead to aggressive proliferation and migration, as well as inhibition of apoptosis through enhancing the activities of PI3K/AKT/mTOR signaling pathway [16]. In vitro study has shown that the expression of Per3 was decreased in CRC, moreover, it played a suppressive role in malignant biological behaviors of the cancer cells [17]. In CCA, previous tumor investigations have reported that cicadian clock disruption induced by abnormal expression of clock genes could signi cantly promote liver carcinogensis [18]. However, the function of Per genes in CCA had been rarely reported.
Per1, a member of the Per subfamily, was proved to play an inhibitory role in several human malignancies via suppressing the biological behaviors of cancer cells [19,20]. Moreover, high expression of Per1 might increase the sensitivity of radiotherapy against tumors, contributing to favorable prognosis of tumor patients [21,22]. In this study, we found that serum level of Per1 was decreased in CCA patients. and its expression pro le showed negative association with malignant clinical parameters. All the data revealed that Per1 as a tumor suppressor gene was involved in initiation and progression of CCA. In addition to CCA, the anti-tumor action of Per1 was also reported in other types of human cancer, such as pancreatic cancer, non-small cell lung cancer, and buccal squamous cell carcinoma [23][24][25]. However, the molecular mechanisms for tumor suppression of Per1 were poorly known in CCA. Further researches were still required.
Given its function in tumor progression, Per1 was considered as a candidate biomarker for tumors. A study of Relles et al. had demonstrated that the expression patterns of Per1 in comparing pancreatic cancer tissues might be a predictive biomarker for survival of the patients [26]. Hsu et al. found that the blood level of Per1 was closely correlated with postoperative survival of patients with head and neck squamous cell carcinoma, suggesting its potential as a non-invasive biomarker for cancers [22]. In the present study, we estimated the value of serum Per1 as a non-invasive tool for early screening of CCA. We found that serum Per1 could be an effective biomarker for CCA patients with high sensitivity and speci city. Circulating Per1 might be a reliable biomarker for early detection and monitoring of CCA. However, the sample size was relatively small in the current study. The clinical signi cance of serum Per1 for CCA diagnosis was needed further veri cation.

Conclusions
In conclusion, Per1 is down-regulated in CCA and negatively correlated with the progression of this disease. What's more, serum Per1 may be a promising biomarker for early diagnosis of CCA. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study. The relative mRNA levels of Per1 in serum of CCA patients and healthy controls. Compared with healthy individuals, serum Per1 level was signi cantly down-regulated in CCA patients (P<0.001). *** indicated P less than 0.001.