Clinical competence of serum miR-372 expression in diagnosing prostate cancer

Background: Prostate cancer (PCa) is considered as a serious healthy burden among males around the world. The limited diagnosis leads to poor outcomes of this malignancy. Aberrant expression of microRNAs (miRNAs) have been found in human cancers and play crucial roles in these malignant diseases. MicroRNA-372 (miR-372), as a type of miRNAs, has ever been investigated in various cancers. The purpose of the present study was to assess the expression patterns and diagnostic value of miR-372 in patients with PCa. Methods: 134 PCa patients and 68 healthy individuals were included in this study. The serum expression of miR-372 was examined by quantitative real-time PCR (qRT-PCR). To verify the diagnostic signicance of miR-372, receiver operating characteristics (ROC) analysis was conducted for PCa patients. Results: Downregulated miR-372 expression was detected in serum samples collected from PCa patients compared with the healthy controls (P<0.001). The decreased expression of miR-372 was inuenced by the positive lymph node metastasis (P=0.016), high prostate-specic antigen (PSA) concentration (P=0.002) and advanced TNM stage (P=0.013). The ROC curve was obtained with an AUC value of 0.896. At the cutoff value of 2.855, the sensitivity and specicity were 82.8% and 87.3%, respectively, suggesting the high diagnostic accuracy of miR-372. Conclusion: In summary, serum downregulated expression of miR-372 could serve as an ecient diagnostic biomarker for patients with PCa.


Background
Prostate cancer (PCa) ranks the second leading cause of deaths due to malignancy for males in Western countries and United State [1]. It is considered as the rst common type among all the tumors diagnosed for men with an increasing incidence and mortality in recent decades [2]. The abundant new diagnosed PCa individuals have poor quality of life [3]. Given the pain from PCa, various strategies have been conducted for the cancer treatment. Prostate-speci c antigen (PSA) testing is considered as the most useful method to diagnose PCa till now [4]. However, the limited sensitivity and speci city for this method leads to controversy for the nonmalignant prostate diseases also perform high PSA concentration [5].
Besides, the inappropriate indication on aggressiveness usually causes the over treatment for some patients with PCa [6]. Considering total of the challenges in the diagnosis and treatment, focused efforts should been taken out on the identi cation of novel and more sensitive and speci c biomarkers to meet the clinical requirements for PCa treatment.
Accumulated evidences demonstrate that various processes are involved in the tumorigenesis and progression of human cancers, including abundant genetic mutations, abnormal expression of genes and aberrant microRNAs (miRNAs) expression [7]. MiRNAs, widespread in eukaryotic cells, represent a series of endogenous single stranded small RNA molecules with approximately 22 nucleotides in length. A wide of clinical processes are in uenced by miRNAs, such as differentiation, cell cycle, proliferation and apoptosis [8]. Aberrant miRNAs expression levels are detected in lots of cancers including PCa, which have been proved to be involved in the tumor progression by regulating the expression of tumor oncogenes and suppressors [9][10][11][12]. As a member of the miRNAs, microRNA-372 (miR-372) expression dysregulation has also been found in some malignancies, such as colorectal carcinoma and testicular germ cell cancer [13,14]. The downregulated miR-372 was detected in PCa serum samples, and was demonstrated to inhibit the migration and invasion of cancer cells in the study by Kong and his colleagues [15]. However, whether miR-372 could perform any clinical signi cance for diagnosis of PCa has never been discussed.
In the present study, the diagnostic value of miR-372 was evaluated and its serum patterns was also investigated in patients with PCa.

Patients and serum specimens
Total of the protocols in our study were approved by the Ethics Committee of Zhongnan Hospital of Wuhan University, and the written informed consents were all obtained from the participators and their families. This study include 134 PCa patients and 68 age and gender matched healthy individuals. The patients were all diagnosed with PCa in Zhongnan Hospital of Wuhan University with no history of other malignancies and had never received any therapy prior to sample collection. 68 healthy volunteers who received the routine physical examination in the hospital without any history of cancers were recruited as controls in this study. The venous blood samples were collected from the patients and healthy controls and stored in EDTA tubes. Serum specimens were obtained by using centrifugation from the blood samples and kept at -80 ℃ for subsequent utilization. The clinical data of patients were recorded in Table 1. The clinicopathological features included age, prostate size, Gleason score, lymph node metastasis, pathological stage, PSA concentration and TNM stage. PSA, prostate-speci c antigen.

RNA extraction
Total RNAs including miRNAs were isolated from the serum specimens using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as per the instruction. To con rm the purity of RNA, NanoDrop ND-1000 (NanoDrop, Wilmington, DE) was used to measure the absorbance at 260 nm and 280 nm, and the ratio of OD A260/A280 was calculated. Only the RNA with the ratio of close to 2.0 was permitted to be used in the subsequent experiments.
Quantitative real-time PCR (qRT-PCR) The revers transcription was carried out to obtain the single stranded cDNA from RNA samples with the application of Transcriptor First Strand cDNA Symthesis Kit (Roche, Vilvoord, Brussel, Belgium).
Expression of miR-372 was examined by qRT-PCR, which was conducted with the SYBR green I Master Mix kit (Invitrogen). The obtained cDNA was ampli ed in these reactions in 7300 Real-Time PCR System (Applied Biosystems, USA). U6 was adopted to act as the endogenous control gene to normalize the relative expression of miR-372. The nal miR-372 expression levels were computed with method of 2 −ΔΔCt .

Statistical analysis
The comparison of miR-372 expression levels between PCa patients and healthy controls was performed by Student's t-test. Chi-square test was adopted to analyze the relationship between miR-372 expression and clinicopathological characteristics of PCa patients. The diagnostic performance of miR-372 expression in patients with PCa was assessed with receiver operating characteristics (ROC) analysis. All these statistical analyses above were carried out by SPSS 18.0 software. Results were considered as statistically signi cance when the P value was less than 0.05.

Results
Serum expression patterns of miR-372 The expression of miR-372 levels in 134 PCa patients and 68 healthy volunteers were examined by qRT-PCR. The results showed that miR-372 expression was signi cantly downregulated in PCa serum samples than that in the healthy controls (P < 0.05, Fig. 1). This result suggested the potential tumor suppressor role of miR-372 in PCa patients.

Association of miR-372 expression with clinicopathological features of PCa patients
To verity whether there was involvement for miR-372 with PCa progression, the relationship between miR-372 expression and clinicopathological characteristics of cancer patients was assessed by Chi-square test. The mean miR-372 expression value was used to classify the patients into low miR-372 expression group (n = 75) and high expression group (n = 59). The clinicopathological data shown in Table 1 revealed that there were closely correlation between miR-372 expression and lymph node metastasis (P = 0.016), PSA concentration (P = 0.002) and TNM stage (P = 0.013). However, the other parameters, including age, prostate size, Gleason score and pathological stage, were not found associated with miR-372 expression (all P > 0.05).

Diagnostic performance of miR-372 in patients with PCa
Considering the aberrant expression of miR-372 in PCa serum specimens, the clinical signi cance of miR-372 was also analyzed for cancer patients. In the current study, ROC curve was constructed to con rm the diagnostic value of miR-372. The results shown in Fig. 2 revealed that the AUC value was 0.896 and the sensitivity and speci city were 82.8% and 87.3% at the cutoff value of 2.855. These data indicated that miR-372 could be used as a high sensitive and speci c diagnostic biomarker of PCa patients.

Discussion
PCa represents a serious healthy problem among men, which ranks the most common malignancy diagnosed in males around the world [16]. It is characterized by its high rates of mortality and complex biological heterogeneity [17]. Since the speci c characteristics of PCa, clinical challenges are presented to identify the cancer patients from healthy individuals. Despite the PSA has been widely used in diagnosis and prognosis of PCa, the outcomes of cancer patients remain dismal mainly due to the limitations of the available diagnostic methods [18]. Thus, it is an urgent need to nd novel and e cient diagnostic biomarkers for patients suffering from PCa. Current studies have highlighted the utilization of tumor related molecules for their diagnostic signi cance to improve the diagnosis of diverse cancers, and results in these studies demonstrated the pivotal role of diagnostic biomarker for cancer treatment [19,20].
Among these diagnostic factors, miRNAs are considered as a class of crucial members, which have been investigated in a great deal of malignancies [21]. Previous data revealed that various biological processes, such as proliferation, metastasis, invasion and apoptosis, can be regulated by miRNAs, indicating their potential function in cancer progression [22]. The aberrant expression patterns of miRNAs have been found in lots of cancers, which suggested their role of tumor oncogene or suppressor [23,24].
As a member of miRNAs, miR-372 has ever been assessed in different human cancers [25,26]. For example, miR-372 was demonstrated to be correlated with proliferation and metastasis of hepatocellular carcinoma [27]. Study by Chen and his colleagues revealed that miR-372 could regulate cancer cell invasion and proliferation of glioma according to target PHLPP2 [28]. Given the results of previous studies, rarely data has been reported about clinical signi cance of miR-372 for patients with PCa. Therefore, we aimed at to explore the diagnostic performance of miR-372 via measuring its serum expression levels and various statistical analyses in PCa patients.
In the present study, the expression of miR-372 in the serum specimens collected from the PCa patients were examined by qRT-PCR. Student's t test was adopted to compare the different miR-372 expression between the two groups. According these analyses, the downregulated miR-372 expression was detected in PCa serum specimens than that in the healthy controls. Furthermore, the relationship of miR-372 expression with clinicopathological characteristics of PCa patients was assessed with Chi-square test.
The results revealed that there were closely correlation between miR-372 expression and lymph node metastasis, PSA concentration and TNM stage. Conversely, no in uence was found for other parameters.
The similar data has also been found in the previous study by Kong et al., which also found the downregulated expression of miR-372 in PCa serum samples [15]. Our results might suggested that miR-372 represented a potential tumor suppressor for PCa and was involved in the cancer progression. Since the expression of miR-372 was abnormal in PCa serum and its potential functional role has ever been demonstrated in cancer cells, we further explored its clinicopathological signi cance in diagnosis of PCa through ROC analysis. The results suggested that miR-372 expression was a high sensitive and speci c diagnostic biomarker to distinguish prostate cancer cases from healthy individuals.
Although we have investigated the expression pattern and diagnostic value of miR-372 in PCa patients, more further studies are still needed to con rm our ndings. In the study of Yu et al., they showed that serum or tissue miR-372 levels were signi cantly up-regulated in CRC patients and it could be a noninvasive biomarker for the early detection and prognosis of CRC [29]. Therefore, the role of miR-372 might be varying in different cancers.

Conclusion
Taken together, all data in our study demonstrated the downregulated expression of miR-372 was involved in the progression of PCa, and had high diagnostic value for patients with PCa. The molecular mechanisms of miR-372 acting in PCa remain further studies. This study was supported by the Ethics Committee of Zhongnan Hospital of Wuhan University and also has been carried out in accordance with the World Medical Association Declaration of Helsinki.

Consent for publication
The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.