NUCKS1 Acts As A Promising Novel Bio-Marker in The Prognosis of Patients with Hepatocellular Carcinoma

Background: Nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) was overexpressed in some tumors, including hepatocellular carcinoma (HCC). However, the clinical signicance of NUCKS1 in HCC was still unclear. The aim of this study was to explore the expression and prognostic value of NUCKS1 in HCC. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative mRNA expression of NUCKS1 in HCC tissues and corresponding adjacent normal tissues. The relationship between NUCKS1 expression and clinical characteristics of patients was analyzed by c 2 test. Kaplan-Meier method and cox regression analysis were applied to estimate the prognostic value of NUCKS1 in HCC. Results: Compared with normal tissues, the relative mRNA expression level of NUCKS1 was signicantly up-regulated in HCC tissues (P < 0.001). And high NUCKS1 expression was closely associated with tumor differentiation, TNM stage, vascular invasion and metastasis (P < 0.05). Kaplan-Meier analysis revealed that the overall survival of HCC patients with low expression of NUCKS1 was obviously longer than those with high NUCKS1 expression (log rank test, P = 0.001). NUCKS1 was an independent prognostic factor of HCC patients via univariate and multivariate cox regression analyses. Conclusions: NUCKS1 may be correlated with the progression of HCC and may serve as a potential factor for the prognosis of this disease. 18.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). The data were expressed as mean ± standard deviation (SD). The difference between two groups was evaluated via Student’s t-test. χ 2 test was applied to analyze the relationship between NUCKS1 expression and clinical features of patients. The survival curve was established through Kaplan-Meier analysis with log rank test. Univariate and multivariate cox regression analyses were utilized to estimate the prognostic value of NUCKS1 in HCC patients. P < 0.05 was considered as statistically signicant.


Background
Hepatocellular carcinoma (HCC) is one of the most aggressive fatal malignant tumors worldwide [1]. The incidence and mortality rates of HCC are high in the world [2]. The tumorigenesis and development of HCC include multifaceted causes, such as liver cirrhosis, hepatitis B virus (HBV) and C virus (HCV) infection [3]. Surgical resection is a major therapeutic method for HCC patients, but it is not effective for patients with late stages [4]. Although the treatments and perioperative management had been improved, the prognosis of HCC was still poor with a very low 5-year overall survival rate due to advanced stages, recurrence and metastasis [5,6]. Therefore, nding novel and effective bio-markers for the diagnosis, therapy and prognosis is an urgent need.
Nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1, also named NUCKS) gene, located on human chromosome 1q32.1, encodes a highly phosphorylated and DNA binding protein that also is methylated, ubiquitylated and acetylated [7]. The expression of NUCKS1 is observed in almost all mammalian tissues and NUCKS1 protein is a substrate of some protein kinases in vitro [8,9]. NUCKS1 had been proven to be involved in several cellular processes, including cell proliferation, cell cycle regulation and the activation of various genes during rapid cell growth [10,11]. A large number of studies had demonstrated that the aberrant expression of NUCKS1 was con rmed in a variety types of human cancers and its over-expression was associated with tumor growth and metastasis [12,13]. A study of Cheong et al. mentioned that the expression of NUCKS1 was increased in HCC tissues compared with that in adjacent non-cancerous tissues at protein level [14]. However, little is known about the prognostic value of NUCKS1 in HCC.
In this study, we investigated the clinical signi cance of NUCKS1 in HCC patients through detecting NUCKS1 expression levels in HCC tissues as well as corresponding adjacent normal tissues, and analyzing the correlation between its expression and the progression of HCC patients. Moreover, we also explored whether the elevated NUCKS1 expression could be associated with poor prognosis of HCC patients.

Patients and samples
In this study, we investigated 126 HCC patients who underwent surgery in Harrison international Peace Hospital. The diagnosis of HCC was con rmed by pathological examinations. None of patients had received any therapies before surgery. This study was approved by the Ethical Committee of this hospital and the written informed consents were obtained from all participators in advance.
HCC tissues and corresponding adjacent normal tissues were collected from 126 patients enrolled in this study. The tissue samples were immediately frozen in liquid nitrogen and store at − 80 ℃ for use. The detail clinicopathological characteristics of HCC patients, including age, gender, liver cirrhosis, tumor size, AFP level, tumor differentiation, TNM stage, vascular invasion and metastasis, were shown in Table 1.
The follow-up was performed with 5 years by a telephone or questionnaires. Patients who died from other diseases or unexpected events should be excluded from our study.

Results
The clinical characteristics of HCC patients As shown in Table 1, there were 44 female and 82 male patients with a mean age of 57.26±15.25 years (range, 34-77 years). Of 126 patients, 87 cases had liver cirrhosis. The sizes of 71 tumors were less than 5 cm and 55 were more than 5 cm. 47 patients had AFP levels less than 20 ng/mL and 79 patients had AFP levels more than 20 ng/mL. Tumor differentiation of 68 cases was well or moderate and 58 cases had poor tumor differentiation. Among these patients, 66 cases were at stage -and 60 cases were at stage -. There were 52 patients of vascular invasion and 45 with metastasis.
The relative expression of NUCKS1 was up-regulated in HCC patients QRT-PCR was used to detect the relative mRNA expression levels of NUCKS1 in HCC tissues and adjacent normal tissues. The result showed that NUCKS1 expression was signi cantly increased in HCC tissues compared to that in corresponding adjacent normal tissues at mRNA level (P < 0.001, Figure 1). Association between NUCKS1 expression and clinical characteristics of HCC patients To explore the role of NUCKS1 in HCC, 126 patients were divided into two groups (high NUCKS1 expression group and low NUCKS1 expression group) according to mean expression of NUCKS1. Then the relationship between NUCKS1 expression and clinical features of patients was analyzed. Elevated NUCKS1 expression was closely associated with tumor differentiation (P = 0.039), TNM stage (P = 0.014), vascular invasion (P = 0.046) and metastasis (P = 0.035) ( Table 1). However, there was no obvious correlation between NUCKS1 expression and age, gender, liver cirrhosis, tumor size or AFP level (P > 0.05, Table 1).

Discussion
In the present study, we detected the relative mRNA expression levels of NUCKS1 in HCC tissues and adjacent normal tissue samples. The result of qRT-PCR showed that NUCKS1 expression was signi cantly higher in HCC tissues than that in normal tissues. In addition, the increased NUCKS1 expression was tightly associated with some clinical factors, including well/moderate tumor differentiation, advanced TNM stage, presence of vascular invasion and positive metastasis of patients. This results revealed that NUCKS1 functioned as an oncogene in HCC, and was involved in carcinogenesis and tumor progression.
NUCKS1 protein is identi ed as one of the cell cycle-related proteins and participates in the response of DNA damage induction [8,15]. Previous studies had reported that NUCKS1 played a crucial role in the regulation of glucose homoeostasis and insulin signalling [16,17]. Besides, NUCKS1 had been shown to be a key transcriptional regulator and chromatin modi er and it could be a potential bio-marker for human diseases, including cancers and metabolic diseases [18]. Similar to the high-mobility group (HMG) proteins, NUCKS1 belonged to the most modi ed proteins in the proteome of mammals and was associated with the progression and diagnosis of human cancer [19]. A study carried out by Kikuchi et al. demonstrated that the mRNA and protein expression levels of NUCKS1 were signi cantly up-regulated in colorectal cancer (CRC) tissues compared to that in normal tissues [20]. A notable correlation was found between NUCKS1 over-expression and invasion, TNM stage and metastasis of CRC patients. In addition, Liu et al. revealed that increased NUCKS1 expression was strongly correlated with stage, histological grade and metastasis of endometrial cancer [21]. Our results were consistent with all the previous studies.
Due to frequent recurrence and metastasis, the prognosis of HCC remains undesirable. Commonly, serum AFP level is considered as a diagnostic and prognostic factor of HCC patients [22]. Moreover, TNM stage and metastasis are both associated with the prognosis of HCC patients. However, they lack accuracy [23,24]. Accumulated evidences had supported that increased NUCKS1 was linked with poor prognosis of human cancers [20,25]. However, its prognostic value in HCC remains poorly known. In current study, Kaplan-Meier survival analysis result showed a negative relationship between NUCKS1 expression and overall survival of HCC patients. Moreover, cox regression analysis further demonstrated that the NUCKS1 expression level was an independent prognostic factor for the survival of HCC patients. Besides, TNM stage and metastasis both were independent factors.
There were still some limitations in our study. Firstly, high expression of NUCKS1 was indicated to be associated with tumor size of patients with cervical squamous cell carcinoma [26], which was inconsistent with our ndings. The functions of NUCKS1 in different tumors were varied and the sample size as well as its source might also cause differences. Next, the precise molecular mechanism of NUCKS1 in the regulation of HCC is still unclear. Thus, given these constraints, further studies are needed to gain insight into the functions of NUCKS1 in the progression of HCC.

Conclusions
In conclusion, the mRNA expression of NUCKS1 is up-regulated in HCC and associated with the progression of this tumor. Besides, NUCKS1 may be a potential bio-marker for the prognosis of HCC. 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. The relative mRNA expression of NUCKS1 in HCC tissues and corresponding adjacent normal tissues.
Compared with normal tissues, NUCKS1 expression was obviously up-regulated in HCC tissues at mRNA level (P < 0.001). *** indicated P less than 0.001.