HTATIP2 Represents an Innovative Bio-marker in Hepatocellular Carcinoma Detection


 Background: This study was designed to investigate the serum level of HIV-1 Tat interactive protein 2 (HTATIP2) mRNA in hepatocellular carcinoma (HCC) patients and its diagnostic significance in the disease.Methods: The serum HTATIP2 mRNA level was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between HTATIP2 expression and clinical parameters was analyzed using Chi-square test. Receiver operating characteristics (ROC) curve was adopted to estimate the diagnostic role of serum HTATIP2 in HCC.Results: HCC patients showed a significantly lower serum level of HTATIP2 than the healthy control (P<0.001). The level of HTATIP2 was closely associated with venous invasion (P=0.011), lymph node metastasis (P=0.007) and TNM stage (P=0.016). ROC curve demonstrated that HTATIP2 could discriminate between HCC patients and healthy individuals at the optimal cutoff point of 2.39. Besides, the AUC was 0.892, with the corresponding sensitivity and specificity of 83.90% and 84.37%, respectively. Conclusions: HTATIP2 is negatively expressed in HCC and may be a diagnostic biomarker for this disease.


Background
Hepatocellular carcinoma (HCC) is one of the most frequently diagnosed liver cancers, with severe mortality [1,2]. There are several well known risk factors for HCC, such as alcohol abuse, hepatitis C virus (HCV), hepatitis (HBV) and fatty liver [3]. Until now, a variety of therapeutic strategies are available for HCC patients, such as surgical resection, local ablation therapy, chemoembolization, liver transplantation and molecular target therapy [4,5]. Despite slight improvements in clinical outcomes, the ve-year survival of HCC patients is still dismal [6]. The low survival rate may be attributed to delay in diagnosis [7]. Unfortunately, the majority of HCC patients are diagnosed with advanced stages owing to the lack of e cient screening methods [8,9]. Therefore, it is urgently needed to investigate novel and effective diagnostic biomarkers for HCC to improve the prognosis of the patients.
HIV-1 Tat interactive protein 2 (HTATIP2), also known as TIP30 or CC3, is located on human chromosome 11p15.1, and codes a 30kD human cellular protein [10,11]. The gene is initially identi ed as a metastasis suppressor in human variant small cell lung carcinoma (v-SCLC) [12,13]. HTATIP2 is ubiquitously expressed in normal tissues, whereas its expression patterns are aberrant in diseases tissues. An increasing number of studies have demonstrated that HTATIP2 acts as a tumor suppressor in various cancers via regulating a series of biological processes, including cell growth, apoptosis, and angiogenesis [14][15][16]. For instances, in pancreatic cancer, down-regulated HTATIP2 expression could facilitate cancer cell invasion and metastasis via controlling the expression of Snail family members [17]. In esophageal squamous cell carcinoma, inhibiting the expression of HTATIP2 was able to active EMT, thus promoting migrative and invasive abilities of the cancer cells [18]. The anti-tumor action of HTATIP2 in HCC was also reported in the previous tumor investigations. The study carried out by Lu et al. reported that upregulation of HTATIP2 through Aspirin treatment was able to weaken invasiveness of HCC cells [19]. However, the diagnostic performance of HTATIP2 in HCC remained poorly known.
In the present study, we examined the serum level of HTATIP2 in HCC patients and explored its diagnostic roles in this disease.

Methods
Patients and serum specimens A total of 118 patients who were pathologically diagnosed with HCC were recruited from Harrison international Peace Hospital. The clinical features of patients were listed in Table 1. All the patients were classi ed based on the World Health Organization (WHO). None of the patients had received any treatments, such as radio-or chemo-therapy. In addition, 64 healthy donors who were age and gender matched with the patients were enrolled in this study. 5 mL peripheral blood was collected from each participant on the morning after fasting for 8-10h. The serum samples were prepared from peripheral blood through centrifugation and stored in EDTA-containing tubes at -80℃. This study was approved by the Ethics Committee of Harrison international Peace Hospital. Each participant signed the informed consent before blood collection.

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from serum samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacture's instructions. Then the total RNA was used to synthesis the rst chain of cDNA by PrimerScript RT reagent kit (Takara, China). QRT-PCR was applied to detect the relative mRNA level of HTATIP2 in serum specimens. The reaction was carried out by SYBR Premix Ex Taq (Takara, China) in the 7500 Real Time PCR system (Applied Biosystems) under optimal conditions. All assays were conducted in triple. β-actin was taken as an internal reference. The primer sequences were as follows: HTATIP2, forward 5'-TCACCTTCGACGAGGAAGCT-3', reverse 5'-GCTCTGCAGACTTCAGACCA-3'; βactin, forward 5'-CGTGGACATCCGTAAAGACC-3', reverse 5'-ACATCTGCTGGAAGGTGGAC-3'. The expression of HTATIP2 mRNA was calculated using the 2 -∆∆Ct method.

Statistical analysis
All data were statistically analyzed by SPSS 18.0 software and Graphpad Prism 5.0. Student's t-test was adopted to evaluate the expression difference of HTATIP2 mRNA between HCC patients and healthy controls. The associations between HTATIP2 expression and clinical parameters of patients were determined by Chi-square test. Receiver operating characteristics (ROC) curve was generated to estimate the diagnostic sensitivity and speci city of HTATIP2 in HCC. P<0.05 was considered signi cant.

Results
Expression of HTATIP2 mRNA was decreased in HCC serum To determine the expression of HTATIP2 mRNA in HCC patients, we conducted the qRT-PCR test. As shown in Figure 1, the expression of HTATIP2 mRNA in HCC serum samples was signi cantly lower than that in healthy controls (1.83±0.59 vs 3.12±0.80, P<0.0001).

Association between HTATIP2 expression and clinical characteristics
The patients were divided into high expression group (n=39) and low expression group (n=79), based on their median expression level of HTATIP2. Chi-square test was applied to estimate the association between HTATIP2 mRNA level and clinical characteristics of the patients. As listed in respectively. Besides, the cut-off value of HTATIP2 for HCC diagnosis was 2.39. All these results indicated that serum HTATIP2 might be a potential diagnostic biomarker in HCC ( Figure 2).

Discussion
HCC is a prevalent malignancy with insidious onset. Tumor stage at diagnosis and appropriate therapy are key for outcomes of HCC patients [7]. To data, there is no available tool for early detection of the disease, due to the unclear etiology and pathogenesis [20]. Consequently, most of the patients are diagnosed at an advanced stage, resulting in low survival rate. The detection of biomarkers in body tissues or uids are considered as a promising way to improve the diagnostic accuracy of HCC [21]. Serum AFP (alpha fetoprotein) is one of the major biomarkers for HCC diagnosis currently. Despite of high speci city, the low sensitivity of AFP may lead to missed diagnosis [22]. Hence, more speci c and sensitivity tumor markers are urgently needed for HCC research.
HTATIP2 is a tumor metastasis-related gene which is primitively discovery by Shtivelman et al. [23]. HTATIP2 plays inhibitory roles in tumorigenesis via regulating various processes, such as inhibiting tumor growth, inducting cancer cell apoptosis, weakening athletic ability, and so on [14][15][16]. As a tumor suppressor gene, HTATIP2 has been reported to be involved in the development and progression of various caners. Chen et al. reported that promoter methylation caused down-regulation of HTATIP2 that contributed to tumor progression of colorectal cancer [24]. Tong et al. suggested that HTATIP2 was downregulated in lung cancer and its decreased expression showed signi cant association with metastatic potential of the disease [25]. In this study, we determined the expression pattern of HTATIP2 in HCC, as well as its functional roles in tumor progression.
In the current study, we found that HTATIP2 mRNA level was down-regulated in HCC patients compared with the healthy controls. Besides, chi-square test demonstrated that the reduced expression of HTATIP2 might contribute to positive venous invasion, lymph node metastasis and advanced TNM stage. The above ndings hinted that HTATIP2, as a tumor suppressor, was involved in the development and progression of HCC. The conclusion was consistent with the previous studies. A related study carried out by Zhu et al. demonstrated that HTATIP2 was decreased in HCC patients and its expression level showed close association with E-cadherin expression. Cell experiments proved that down-regulated HTATIP2 expression contributed to activated EMT, as well as enhanced invasion and motility of HCC cells, suggesting the inhibitory function of the gene in malignant development and progression of HCC [26].
As an important tumor suppressor, HTATIP2 was proved to play a predictive role in tumor initiation and progression. In pancreatic ductal adenocarcinoma, decreased expression of HTATIP2 predicted poor prognosis for the patients [17]. Among gastric cancer patients, reduced expression of HTATIP2 represented dismal overall survival [27]. In the present study, we evaluated the diagnostic value of HTATIP2 in HCC. ROC curve demonstrated that HTATIP2 could discriminate between HCC patients and healthy controls with satisfactory sensitivity and speci city. Though we had con rmed the low expression of HTATIP2 in HCC patients and identi ed its diagnostic role in this malignancy, its precise mechanism on this disease was still not well understood. Accumulating evidences proved that HTATIP2 might be linked with the development and progression of angiogenesis in HCC, which was realized through modulating the transcription of angiogenesis regulator [28]. Besides, it was also proposed that HTATIP2 served as a tumor suppressor via inducing cell apoptosis in HCC [29]. All the related researches could provide theoretical foundations for our further studies.

Conclusion
In conclusion, HTATIP2 expression is signi cantly lower in HCC patients than that in the healthy controls.
The expression level of HTATIP2 may e ciently discriminate HCC patients from healthy controls. 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.
Competing interestsThe authors declare that they have no competing interests.
Funding Not applicable.  The mRNA level of serum HTATIP2 in HCC patients and healthy controls was determined using the qRT-PCR. It was found that HTATIP2 was up-regulated in HCC patients compared with healthy control. ***: suggested P<0.0001.