Relationship of Common Variants in PRL-3 Gene With Susceptibility and Prognosis of Prostate Cancer

Background: Phosphatase of regenerating liver-3 (PRL-3) was involved in a variety of malignancies. In this study, we investigated the expression pattern of PRL-3 and its prognostic signicance in prostate cancer (PCa). Methods: The expression proles of PRL-3 in 126 pairs PCa tissues and adjacent normal specimens were analyzed by quantitative rreal-time polymerase chain reaction (qRT-PCR). Chi-square test was used to estimate the association of PRL-3 level with clinicopathological factors. Survival analysis was performed by Kaplan-Meier method with log rank test. Prognosis analysis was carried out using Cox proportional hazard models. Results: PRL-3 expression was signicantly up-regulated in PCa tissues compared to that in normal controls (P<0.001). Moreover, the expression of PRL-3 was positively associated with lymph node metastasis (P=0.038), Gleason score (P=0.003) and lymphovascular invasion (P=0.013). High PRL-3 expression predicted poor overall survival (OS) (log rank test, P=0.019) and dismal BCR-free survival (log rank test, P=0.021) for PCa patients. PRL-3 was an independent prognostic factor for OS (HR=2.619, 95%CI=1.129-6.076, P=0.025) and BCR-free survival (HR=2.120, 95%CI=1.095-4.105, P=0.026) in PCa. Conclusions: PRL-3 expression is elevated in PCa, and exhibits positive association with malignant tumor progression. PRL-3 may serve as a prognostic biomarker for patients with PCa.

Accumulating evidences suggest that PRL-3 is involved in multiple steps in tumorigenesis, including cell proliferation, motility, metastasis, and invasion [12,13]. High expression of PRL-3 may enhance the malignant potential of the cancer cell through various pathways, such as Ras/MAPK, PI3K/Akt and SRC [14][15][16]. The oncogenic roles of PRL-3 has been reported in colon cancer [17], gastric cancer [12], breast cancer [18], etc. In PCa, the expression of PRL-3 showed increased in diseased tissues, and was positively correlated with proliferative and athletic abilities of the cancer cells [19]. Based on the related researches, we deduced that PRL-3 might be a credible biomarker for prognosis prediction in PCa.
In this study, the expression pattern of PRL-3 mRNA in PCa tissue specimen was detected, as well as its association with clinical characteristics. In addition, we evaluated the effects of PRL-3 expression on overall survival of PCa patients, and its prognostic value in the disease.

Patients and specimens
All the experiments were performed in accordance with ethical and legal standards, and the approval of this study protocol was obtained from the Ethics Committee of Harrison International Peace Hospital. The PCa tissues and matched adjacent normal specimens were obtained from 126 PCa cases who had underwent radical prostatectomy in Harrison International Peace Hospital. Before surgery, none of the patients had received chemotherapy, radiation therapy or androgen deprivation treatment. After prostatectomy, the tissues samples were immediately frozen in liquid nitrogen and stored at -80˚C until use. The follow up was conducted from 2 months to 60 months. Written informed consent was obtained from all patients, and the clinical information of the patients was shown in Table 1. . GAPDH was used as internal control. The sequences of used primers were listed: PRL-3 forward 5'-AGTTGCCCGCTTTACTTTGGTTGG-3' and reverse 5'-AGGAAGCTGCCCACTGTTTGGATA-3'. GAPDH forward 5'-CGGAGTCAACGGATTTGGTCGTAT-3', reverse 5'-AGCCTTCTCCATGGTGGTGAAGAC-3'. The relative expression of PRL-3 was calculated by 2 −ΔΔCt . All experiments were in triplicate.

Serum PSA measurements
After prostatectomy, serum prostate-speci c antigen (PSA) level of patients were measured by ELISA kit.
Moreover, the PSA level > 0.2 ng/ml was de ned as biochemical recurrence (BCR).
Statistical analysis SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 (GraphPad, San Diego, CA, USA) were used for statistical analysis. The expression value of PRL-3 was expressed as mean ± SD, and its difference. between two groups were compared by Student's t-test. Chi-square test was used to evaluate the association between PRL-3 expression and clinicopathological variables of the patients. Survival analysis was estimated using the Kaplan-Meier method with the log-rank test, and Cox's regression model was applied for prognosis analysis. P < 0.05 was considered statistically signi cant.

PRL-3 expression is increased in PCa
To determine the potential role of PRL-3 in the tumorigenesis and progression of PCa, we assessed PRL-3 mRNA expression in 126 PCa tissues and matched adjacent normal tissues specimens by qRT-PCR. As shown in Fig. 1, the expression of PRL-3 in PCa samples was signi cant higher than that in normal controls (P < 0.001).

Associations between PRL-3 expression and clinicopathological parameters
In this study, the mean expression value of PRL-3 mRNA in 126 PCa patients was used as the cutoff to divide the cases into high expression (n = 60) and low expression (n = 66) groups. Chi-square test indicated that the increased expression of PRL-3 was signi cantly associated with positive lymph node metastasis (P = 0.038), high Gleason score (P = 0.003) and lymphovascular invasion (P = 0.013).
However, no association between expression levels of PRL-3 with age, perineural invasion or surgical margin (all, P > 0.05) was found (Table 1) The association of PRL-3 with the overall survival and BCR-free survival Kaplan-Meier survival curves were constructed to evaluate the effects of PRL-3 expression on overall survival and BCR-free survival of patients with PCa. The curves suggested that patients with high PRL-3 expression had shorter overall survival than those with low expression (log rank test, P = 0.019; Fig. 2). The BCR-free survival curves demonstrated that high expression of PRL-3 predicted poor BCR-free survival for patients with the disease (log rank test, P = 0.021; Fig. 3).
More importantly, the univariate and multivariate Cox proportional hazards model analysis was applied to evaluate the predictive signi cance of clinical parameters and PRL-3 expression for overall survival and BCR-free survival. Analysis results revealed that lymph node metastasis (P = 0.030), Gleason score (P = 0.028) and PRL-3 expression (P = 0.025) were signi cantly correlated with overall survival of PCa patients. Multivariate analysis demonstrated that increased PRL-3 expression was an independent biomarker for poor prognosis in PCa (HR = 2.619, 95%CI = 1.129-6.076, P = 0.025) ( Table 2). Likewise, cox analysis for BCR-free survival revealed that upregulation of PRL-3 might be employed as a signi cant predictor for dismal BCR-free survival (HR = 2.120, 95%CI = 1.095-4.105, P = 0.026)( Table 3).

Discussion
Prostate cancer (PCa) is a frequently diagnosed malignancy in males worldwide, with severe mortality [20]. Although the diagnosis and therapies for PCa have been signi cantly improved, the clinical outcome of the patients is still poor. Recurrence and metastasis are the major reasons for cancer-related deaths in PCa patients [4]. Up to now, serum PSA is the only biomarker for early screening and monitoring of PCa in clinical setting [21]. Despite of the high sensitivity, the low speci city of PSA may lead to over-treatments [22]. Therefore, it is of great importance to develop reliable biomarkers for prognosis estimation and guidance of treatments inPCa. In the present study, our results con rmed that the expression of PRL-3 was remarkably increased in PCa tissues, and signi cantly correlated with tumor progression and outcomes of the patients, suggesting its possibility to act as an independent prognostic biomarker for patients with PCa.
The PRL family, comprised of PRL-1, PRL-2, and PRL-3, plays an important role in development and metastasis of human malignancies, based on their regulatory roles in cellular processes [23]. The family member of PRL may control cell growth and tumorigensis through various pathways. For instance, the study carried out by Jin et al. demonstrated that the oncogenic function of PRL-1 in progression and metastasis of hepatocellular carcinoma might be mediated by PI3K/AKT/GSK3β signaling pathway. Upregulation of PRL-1 predicted poor prognosis for patients with the disease [24]. As a member of PRL subgroup, PRL-3 also played a promoting role in malignant progression of cancer. Xiong et al. suggested that the expression of PRL-3 showed increased in metastatic gastric cancer tissues, and it might control the aggressive potential of the cancer cell via activating the PI3K/Akt signaling pathway, and upregulating MMP-2/MMP-9 expression [12]. Besides of gastric cancer, the over-expression of PRL-3 was also observed in ovarian cancer, breast cancer, esophageal squamous cell carcinoma, cervical carcinoma, and colorectal cancer [25][26][27][28][29]. The dysregulation of PRL-3 in tumorigenesis showed signi cant association with tumor progression and clinical outcomes of the patients which might be employed as a prognostic biomarker for the cancers. However, the prognostic performance of PRL-3 in PCa remained unclear.
In the current study, we investigated the expression pattern of PRL-3 in PCa tissues specimens, as well as its association with clinical parameters of the patients. Analysis results suggested that PCa tissues exhibited up-regulated expression of PRL-3, compared with non-cancerous tissues. Moreover, the elevated expression of PRL-3 was positively correlated with lymph node metastasis, Gleason score and lymphovascular invasion. The data revealed that PRL-3 served as an oncogene in progression of PCa. The increased expression of PRL-3 might enhance the malignant development and progression of the disease. It was reported that the over-expression of PRL-3 might promote the growth and migration of PCa cell line, thus contributing to the pathogenesis of PCa [19]. However, the molecular mechanisms underlying the oncogenic action of PRL-3 in PCa were poorly known. Further analysis was still required to address the issue.
Growing evidences have reported hat PRL-3 was an useful indicator for patient outcome in several human cancers, including hepatocellular cancer [25], gastric cancer [30] and colorectal cancer [31]. In the present study, survival analysis showed that patients with high PRL-3 expression had signi cantly worse OS and BCR-free survival than those with low PRL-3 expression. More importantly, multivariate analysis demonstrated PRL-3 could serve as an independent biomarker for clinical outcomes and BCR-free survival for patients with PCa. However, the results might be limited by the single-center study design and relatively small sample size in the current study. The predictive value of PRL-3 for prognosis of PCa was required to be identi ed by further studies.

Conclusions
In summary, PRL-3 expression is signi cantly upregulated in PCa tissues, and positively correlated with aggressive development and progression of the disease. PCa may be an independent prognostic factor for patients with PCa. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Abbreviations
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.  Associations between PRL-3 expression and survival of patients after prostatectomy. Patients with high PRL-3 expression showed signi cantly shorter overall survival than those with low PRL-3 expression (log rank test, P=0.019).