Rab25 Acts as a Promising Novel Bio-Marker in the Prognosis of Patients with Hepatocellular Carcinoma

Background: Rab25 was indicated to be involved in several human tumors. However, the clinical signicance of Rab25 in hepatocellular carcinoma (HCC) was still unclear. The purpose of this study was to investigate the expression and prognostic value of Rab25 in HCC. Methods: The relative mRNA expression levels of Rab25 in HCC tissues and adjacent normal tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Chi-square test was used to analyze the relationship between Rab25 expression and clinical characteristics of patients. The prognostic value of Rab25 in HCC was estimated through Kaplan-Meier method and cox regression analysis. Results: Rab25 gene expression level was signicantly higher in HCC tissues than that in normal tissues (P<0.001). Importantly, the increased Rab25 expression was closely associated with TNM stage (P=0.024), metastasis (P=0.022) and invasion classication (P=0.039). Moreover, patients with high Rab25 expression tended to have obviously shorter overall survival than those with low expression of Rab25 (log rank test, P<0.001) via Kaplan-Meier analysis. Univariate and multivariate cox regression analyses revealed that Rab25 was an independent prognostic factor of HCC. Conclusions: Rab25 is up-regulated in HCC and contributes to the progression of this tumor. What’s more, Rab25 may be a potential bio-marker for the prognosis of HCC.


Background
Currently, hepatocellular carcinoma (HCC) is a most frequent malignancy worldwide [1]. HCC is one of the major causes of incidence and mortality rates among malignant tumors [2]. The risk factors for the progression of HCC consist of chronic hepatitis virus infection and liver cirrhosis [3]. Although surgical resection is the most common therapies for HCC, the death rate is still high due to the frequent recurrence and high metastasis [4]. In addition, most HCC patients are diagnosed with advanced stages and the current treatments are not effective for them [5]. Because of the above-mentioned reasons, the prognosis of HCC is poor and the 5-year overall survival is very low [6]. Therefore, it is important to nd novel biomarkers for the prognosis and targets for the therapy of HCC.
Rab GTPases family plays crucial roles in regulating vesicle transportation [7]. Previous studies had demonstrated that several members of Rab GTPases were involved in some processes, including the progression of human tumors [8,9]. Rab25 is a member of the Rab11 family and located on human chromosome 1q22. Rab25 gene encodes an intracellular transport protein that participates in the regulation of signal transduction and pathways, including cell proliferation, differentiation and migration of cancers [10]. The expression of Rab25 was considered to be ubiquitous and enriched in epithelial cells. A growing body of evidence had reported that the aberrant expression of Rab25 was con rmed in a variety types of tumors [11,12]. In addition, a study suggested that knockdown of Rab25 could suppress cell growth, cell cycle progression and induce cell apoptosis of ovarian cancer [13]. Moreover, Rab25 had been proven to be up-regulated in HCC, and its over-expression contributed to tumor cell proliferation as well as invasion [14]. However, little is known about the prognostic value of Rab25 in HCC.
In this study, we detected the relative mRNA expression levels of Rab25 in HCC tissues and corresponding adjacent normal tissues. The relationship between Rab25 expression and clinical characteristics of patients was analyzed. The prognostic value of Rab25 was also assessed.

Patients and samples
We recruited 132 HCC patients who underwent curative resection at Cancer Hospital ,Chinese Academy of Medical Sciences & Peking Union Medical College. None of the patients had received any treatments before surgery. Histopathological diagnosis was con rmed by the pathologists with extensive experience.
This study was approved by the Ethical Committee of the hospital and the written informed consents were obtained from all patients in advance.
HCC tissues and corresponding adjacent normal tissues were collected from HCC patients and immediately frozen in liquid nitrogen. The tissue samples were stored at − 80℃ before RNA extraction.
The detail clinicopathological characteristics of HCC patients were shown in Table 1. Patients' follow-up was conducted with 5 years via a telephone or questionnaires. Patients whose deaths were not directly associated with HCC should be excluded from this study. patients had liver cirrhosis and 79 patients had AFP levels less than 20 ng/mL. 74 tumors were less than 5 cm in size and 58 tumors were more than 5 cm in size. There were 68 cases with well or moderate differentiation and 64 cases with poor differentiation. Of 132 patients, 67 patients were at stage -and 65 patients were at stage -. There were 43 patients with positive metastasis and 50 patients of T3 + T4 invasion classi cation.

Rab25 expression was up-regulated in HCC patients
The relative mRNA expression levels of Rab25 in HCC tissues and adjacent normal tissues were detected by qRT-PCR. Compared with normal tissues, Rab25 mRNA expression was signi cantly increased in HCC tissues (P < 0.001, Fig. 1). According to the median Rab25 expression, 132 HCC patients were divided into high Rab25 expression group and low Rab25 expression group.
Relationship between Rab25 expression and clinical features of HCC patients To explore whether Rab25 was involved in the development of HCC, we analyzed the correlation between its expression and clinical characteristics of patients. As shown in Table 1, a notable correlation was found between Rab25 expression and TNM stage (P = 0.024), metastasis (P = 0.022) as well as invasion classi cation (P = 0.039). However, Rab25 expression was not obviously associated with age, gender, liver cirrhosis, AFP level, tumor size or differentiation (P > 0.05, Table 1).

Increased expression of Rab25 was associated with poor prognosis of HCC patients
In Kaplan-Meier analysis, the overall survival time of HCC patients with low Rab25 expression was markedly longer than those with high expression of Rab25 (log rank test, P < 0.001, Fig. 2). To estimate whether Rab25 expression was associated with the prognosis of HCC, cox regression analysis was conducted. Univariate and multivariate analyses using cox regression analysis revealed that Rab25 expression (HR = 2.149, 95%CI = 1.276-3.618, P = 0.004), TNM stage (HR = 2.041, 95%CI = 1.215-3.429, P = 0.007) and metastasis (HR = 1.860, 95%CI = 1.125-3.078, P = 0.016) were all correlated with the overall survival and they might be independent prognostic factors of HCC patients ( Table 2).

Discussion
Rab GTPases family members, belonging to the rat sarcoma (Ras) oncoprotein small GTPases, are key regulators of intracellular vesicle transport that plays an important role in carcinogenesis and tumor progression [7,15]. It reported that Rab GTPases was associated with several tumor-related processes [8].
Similar to other members of Rab GTPases family, Rab25 had been proven to be speci c in epithelial cells and function in various cancers [16]. Previous study had showed that Rab25 could enhance the aggressiveness of tumors through directly interacting with integrin and thereby promoting tumor cell invasion [17]. It also revealed that the aberrant expression of Rab25 might be involved in tumorigenesis, progression and prognosis of neoplasia [18]. However, the clinical signi cance of Rab25 in HCC is still unclear.
In the present study, we evaluated the relative mRNA expression levels of Rab25 in HCC tissues and corresponding adjacent normal tissues. The outcome suggested that Rab25 gene expression was obviously increased in HCC tissue samples compared to that in normal tissues. It revealed that Rab25 might exhibit tissue-speci c function as an oncogene in HCC. This result was accorded with the previous studies. A study of Mitra et al. demonstrated that the mRNA and protein expression levels of Rab25 were signi cantly elevated in breast cancer cell lines [19]. In the study of Sheach et al., tissue microarray section showed the protein expression of Rab25 was higher in ovarian cancer [20].
To explore the effect of Rab25 on the progression of HCC, we analyzed the relationship between Rab25 expression and clinical characteristics of patients. Interestingly, patients with high Rab25 expression tended to have advanced TNM stage, positive metastasis and high invasion classi cation which were aggressive clinical parameters representing advanced progression, invasion and metastasis. Similar ndings were occurred in other cancers. Li et al. mentioned that the increased expression level of Rab25 was closely associated with invasion classi cation, lymph node metastasis and pathological stage of renal cell carcinoma patients [21]. In gastric cancer, elevated Rab25 expression was more often observed in patients with late pathological stage, positive lymph node metastasis and distant metastasis [22].
According to these studies, we hypothesized that Rab25 expression was involved in the development of human tumors, including HCC.
Recently, some clinical factors, such as serum AFP level, TNM stage and metastasis, are shown to be associated with the prognosis of HCC [23]. However, they may not accurately assess the prognosis due to heterogeneity in HCC patients. In this study, Rab25 expression was closely correlated with TNM stage and metastasis, suggesting Rab25 might be linked with the prognosis of HCC. Furthermore, the result of Kaplan-Meier analysis showed that patients with high expression of Rab25 had markedly shorter overall survival than those with low Rab25 expression. Univariate and multivariate cox regression analyses indicated that Rab25 might be an independent prognostic factor of HCC. Similar to our nding, a large number of researches had demonstrated that Rab25 had potency to predict the overall survival and disease-free survival of tumors [24,25]. Moreover, metastasis and TNM stage both were independent factors in this study.

Conclusions
In conclusion, we prove that Rab25 expression is up-regulated in HCC and correlated with tumor progression. Besides, Rab25 may be an independent prognostic factor of HCC. However, considering the constraints of samples size and its source, further studies are needed.

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.

Figure 1
The relative mRNA expression levels of Rab25 in HCC tissues and paired adjacent normal tissues.
Compared with normal tissues, the expression of Rab25 was obviously up-regulated in HCC tissue samples at mRNA level (P<0.001). *** indicated P less than 0.001.

Figure 2
Kaplan-Meier analysis for the overall survival of HCC patients. The overall survival of patients with low expression of Rab25 was signi cantly longer than those with high Rab25 expression (log rank test, P<0.001).