Sperm-Associated Antigen 9 Assumes Signifying Capacity in Hepatocellular Carcinoma Diagnosis

Background: Sperm-associated antigen 9 (SPAG9) has been proposed as a novel biomarker for early diagnosis of human tumors. This study was aimed to assess the clinical value of serum SPAG9 for HCC diagnosis. Methods: Serum SPAG9 was measured by quantitative real-time ploymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Chi-square test was applied to evaluate the association between serum SPAG9 mRNA level and clinical characteristics. The diagnostic accuracy of the serum SPAG9 mRNA was assessed using receiver operating characteristic (ROC) curve. Results: Serum SPAG9 level was signicantly higher in HCC patients than that in healthy controls at both mRNA and protein levels (P<0.01 for all). Furthermore, serum SPAG9 mRNA level was positively correlated with TNM stage (P=0.047), tumor size (P=0.044), and lymph node metastasis (P=0.014). The area under the curve (AUC) of the ROC curve was 0.794, with a sensitivity of 71.4%, a specicity of 80.4%, suggesting the high diagnostic accuracy of serum SPAG9 mRNA for HCC. The cutoff value was 1.030. Conclusions: Serum SPAG9 is signicantly increased in HCC, and positively correlated with aggressive clinical characteristics. SPAG9 may serve as a diagnostic biomarker for HCC.


Background
Hepatocellular carcinoma (HCC) is one of the most common primary liver malignancies, representing a leading cause of cancer-related mortality worldwide [1,2]. The major risk factors for HCC include hepatitis B (HBV) or C (HCV) viral infection, heavy alcohol intake, exposure to a atoxin or vinyl chloride, and chronic liver diseases [3,4]. Despite various available therapeutic approaches for HCC, the ve-year survival of the patients is still unsatis ed, especially those diagnosed with advanced stages [5]. Delay in early diagnosis and recurrence may be responsible for the high mortality [6]. Ultrasonography is a traditional method for HCC diagnosis, but the high cost, low sensitivity and speci city limit its wide application in clinical setting [7]. Serum biomarkers detection may provide a promising approach for screening and surveillance of HCC. Currently, alpha-fetoprotein (AFP) is widely accepted as a gold standard serum marker for the diagnosis of HCC. However, growing evidences have showed that the diagnostic accuracy of AFP is unfavorable, and patients with chronic hepatitis, liver cirrhosis, or other liver diseases also exert elevated serum levels of AFP [8,9]. Thus, it is critical to identify novel and reliable markers to improve the diagnostic accuracy and management of HCC.
Sperm-associated antigen 9 (SPAG9), a novel identi ed member of cancer testis (CT) antigen family, plays important roles in c-Jun NH2-terminal kinase (JNK) signaling pathway via serving as a scaffolding protein [10,11]. SPAG9 is located on human chromosome 17q21, a susceptibility locus for various human cancers [12]. It was reported that abnormal expression of SPAG9 played important roles in cell proliferation, growth, and appoptosis [13]. Accumulating evidences reported that aberration of SPAG9 was observed in various malignancies, such as ovarian cancer, endometrial carcinoma, renal cell carcinoma, breast cancer, non-small cell lung cancer, and human astrocytoma [12][13][14][15][16][17]. SPAG9 has been identi ed as a predictive biomarker for tumors, including breast cancer, cervical cancer, and colorectal cancer [15,18,19]. The function of SPAG9 in HCC was also reported in the previous studies. Yan et al. reported that SPAG9 was up-regulated in HCC tissues, moreover, its over-expression was signi cantly correlated with aggressive potential of the cancer cell lines [20]. Therefore, we deducted that serum SPAG9 might be a potential diagnostic biomarker for HCC.
In the present study, we examined the expression of serum SPAG9 in HCC patients, as well as its association with clinical characteristics. In addition, ROC curve was plotted to evaluate the diagnostic accuracy of serum SPAG9 for HCC.

Methods
Patients and serum samples classi cation. Patients who received preoperative chemotherapy, radiotherapy or immune therapy would be excluded from the study. 96 gender and age matched healthy volunteers who were recruited from the same Hospital at the same period served as controls. From each participant, 5 ml venous blood was obtained in a biochemistry tube. The tubes were centrifuged at 4000 rpm for 10 minutes. Serums were separated from the cellular components of blood, transferred to Eppendorf tubes, and stored at -80℃ for further assay.

RNA extraction and quantitative real-time ploymerase chain reaction (qRT-PCR)
Total RNA was isolated from serum specimens using Trizol reagent (Invitrogen, USA) following the instruction of the manufacture. The quality of the RNA samples were detected by spectrophotomete and 1% agarose gel electrophoresis. Then, total RNA was used for reverse transcribed reaction which was carried out by Prime Script RT reagent kit (Takara, DRR037A, China). The relative mRNA level of SPAG9 was detected by qRT-PCR. The reaction was performed with SYBR Premix Ex Taq (Takara, China) in the Applied Biosystems 7900 Fast Real-Time PCR system (Applied Biosystems, Foster City, California, USA). GAPDH served as internal control, and the primers sequences were as follows: GAPDH forward: 5'-GCCAGGGGTGCTAAGCAG-3', reverse: 5'-GCCAGGGGTGCTAAGCAG-3'; SPAG9 forward: 5'-GACAGAGATGATTCGGGCATCACGAGAAAA-3', reverse: 5'-CTAAGTTGATGACCCATTATTATACCTCGACTG-3'. 2 −ΔΔCt method was used for data analysis.

Enzyme-linked immunosorbent assay (ELISA)
The protein level of SPAG9 was detected by ELISA assay. The assay was performed with human SPAG9 ELISA kit (R&D Systems, USA), and the operations were processed according to the manufacturer's protocol. Each reaction was tested in duplicates, and the mean was used for nal data analysis.

Statistical analysis
All statistical analyses were carried out using SPSS 18.0 statistical software (SPSS Inc., Chicago, USA), and graphs were plotted using GraphPad Prism 5. Continuous variables were presented at mean ± standard deviation (SD), and compared by Student's t test. The association between serum SPAG9 mRNA expression and clinicopathological characteristics was evaluated using chi-square test. Receiver operating characteristic curve (ROC) was conducted to determine the feasibility of serum SPAG9 as a diagnostic marker for HCC. All tests were two-tailed, and P < 0.05 was considered signi cant.

Results
The expression level of serum SPAG9 in HCC patients The mRNA and protein levels of serum SPAG9 was evaluated in 105 HCC patients and 96 healthy controls. Analysis results suggested that serum SPAG9 mRNA expression was signi cantly higher in HCC patients than that in healthy controls (P < 0.001) (Fig. 1).

The correlation between serum SPAG9 expression and clinicopathological characteristics
To better understand the potential roles of serum SPAG9 in HCC development and progression, we further analyzed the potential correlation of serum SPAG9 level with clinicopathological parameters of HCC. In our study, the patients were divided into high expression group (n = 53) and low expression group (n = 52), according to their median mRNA levels of SPAG9. The statistical analysis showed that serum SPAG9 expression was signi cantly associated with TNM stage (P = 0.047), tumor size (P = 0.044), and lymph node metastasis (P = 0.014). No signi cant association was observed between SPAG9 expression and other clinicopathological features, including age, gender, hepatitis, cirrhosis, or tumor differentiation (all P > 0.05) ( Table 1). The diagnostic value of serum SPAG9 in HCC patients ROC curve analysis was carried out to determine the feasibility of serum SPAG9 mRNA as a diagnostic biomarker for HCC. The curve demonstrated that serum SPAG9 mRNA could distinguish the HCC patients from the healthy individuals, with the AUC value of 0.794. The cut-off value of SPAG9 mRNA for HCC diagnosis was 1.030, with the sensitivity of 71.4% and the speci city of 80.4% (Fig. 3) .

Discussion
HCC represents one of the most common and aggressive human malignancies, with severe mortality worldwide. Although various treatment methods are available for HCC, such as surgical resection, liver transplantation, and chemoembolization, the 5-year survival rate has not been signi cantly improved during the past several decades [21]. Like other cancers, early diagnosis is pivotal for prognosis of HCC patients. Currently, ultrasonography combined with serum AFP is accepted as routine approach for screening and surveillance of HCC [22]. However, the low diagnostic accuracy may limited their wide application [7]. Thus, novel and sensitive biomarkers are in urgent need to improve the clinical outcomes of HCC patients. This study was performed to explore valuable non-invasive serum biomarkers for early diagnosis of HCC.
Cancer testis (CT) antigens are not expressed in normal tissues, except the testis, but are abundant in cancerous tissues [23]. The expression of CT antigens family makes its family member hold the potential to serve as a diagnostic biomarker for malignancy. SPAG9, a member of the CT antigens family, is abnormally expressed in various types of cancers. In the current study, we investigated the expression pro le of serum SPAG9 in HCC patients both at mRNA and protein levels. Analysis results demonstrated that both of SPAG9 mRNA and protein levels were increased in HCC patients, compared with healthy individuals. Moreover, the elevated serum SPAG9 mRNA expression was strongly associated with advanced TNM stage, large tumor size, and positive lymph node metastasis. All the data revealed that SPAG9, as a tumor oncogene, played promoting roles in malignant progression of HCC. The conclusion was consistent with the previous studies. The study carried out by Xie et al. demonstrated that the expression of SPAG9 was positive in HCC tissues specimens, moreover, its overexrepssion showed close link with aggressive clinical characteristics and poor prognosis of the patients [24]. Taken together, the expression pattern of SPAG9 was positively correlated with malignant progression of HCC that might be a potential predictive biomarker and therapeutic target for the disease.
Early diagnosis represents a great challenge for HCC, due to the unidenti ed etiology and malignant potential of the cancer [25]. The genetic alterations during tumorigenesis may provide reliable information for tumor initiation, development and progression that are considered as novel indicators for early diagnosis and prognosis estimation in malignancy. In the previous tumor investigations, various molecular biomarkers were con rmed for HCC diagnosis. For instance, Hou et al. reported that the expression of NEU1 (neuraminidase 1) was signi cantly different between HCC samples and normal specimens, moreover, its expression level was signi cantly associated with clinical information of the patients, revealing its diagnostic and prognostic potential for HCC [26]. Lin et al, found that serum miR-224 was up-regulated in patients with early-stage HCC that might be employed as a biomarker for early diagnosis of the disease [27]. Biomarkers detection might be a promising way for early diagnosis of HCC.
In the present study, we investigated the feasibility of serum SPAG9 mRNA used for early diagnosis of HCC. ROC curve demonstrated that serum SPAG9 could distinguish the HCC patients from the healthy individuals with high sensitivity and speci city. Serum SPAG9 might be a potential diagnostic biomarker for HCC.
Although the diagnostic performance of serum SPAG9 was investigated in this study, there were still several limitations. Firstly, the speci c oncogenic mechanism of SPAG9 in HCC remained unidenti ed. Xie et al. reported that overexpression of SPAG9 in human HCC contributed to cancer cell growth through regulation of cyclin proteins, thus promoting malignant progression of the disease [24]. Yan et al. showed that SPAG9 could promote the metastasis of HCC through inducing migration and invasion of the cancer cell lines [20]. Taken together, SPAG9 might be involved in the progression of HCC via controlling the aggressive behaviors of the cancer cell lines. However, the molecular mechanisms for SPAG9 function in HCC remained unclear. Further researches were still required. Second, the sample size was relatively small in the current study. The diagnostic value of serum SPAG9 for HCC in clinic were still needed further investigations.

Conclusions
In summary, SPAG9 is up-regulated in HCC, and positively associated with aggressive clinical characteristics of the patients. Serum SPAG9 may be a potential diagnostic biomarker for HCC.

List Of Abbreviations
Sperm-associated antigen 9 (SPAG9) quantitative real-time ploymerase chain reaction (qRT-PCR) 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 expression of serum SPAG9 mRNA in HCC patients and healthy controls. Serum expression level of SPAG9 in HCC were strongly increased compared with healthy controls (***: suggested P<0.000).

Figure 3
Receiver operating characteristics (ROC) curve was plotted based on serum SPAG9 mRNA level of HCC patients and healthy controls. The area under the receiver characteristics curve (ROC-AUC) was 0.794 (95% con dence interval 0.731-0.856), suggesting serum SPAG9 mRNA could discriminate between HCC patients and healthy individuals. The The best cut-off value was 1.030, and sensitivity and speci city were 71.4% and 80.2% , respectively.