MiR-378 Has the Capacity to Serve as a Diagnostic Biomarker for Prostate Cancer Patient

Background: This study aimed to examine the expression of serum miR-378 in prostate cancer (PCa) patients and healthy individuals and to identify the value of miR-378 in PCa diagnosis. Methods: The expression of serum miR-378 between groups was compared by t-test. The association between miR-378 expression and clinical characteristics of PCa patients was assessed using Chi-square test. The diagnostic value of serum miR-378 in PCa was estimated by the receiver operating characteristic (ROC) analysis. Results: The expression level of serum miR-378 in PCa patients was signicantly lower than that in healthy individuals (P<0.0001). MiR-378 expression was affected by positive AR (P=0.004), large Gleason score (P=0.013) and advanced TNM stage (P=0.020), however, it had no relationship with age, serum PSA, NED rate and urine retention (all, P>0.05). The ROC curve showed that the optimal cutoff value was 1.845, giving the sensitivity and specicity of 75.21% and 89.77%, respectively. Besides, the area under the ROC curve (AUC) was 0.894, indicating serum miR-378 was of great diagnostic value in screening PCa patients from healthy controls (P<0.0001, 95%CI =0.852-0.936). Conclusions: Taken together, the increased expression of serum miR-378 might act as a potential biomarker for PCa diagnosis.


Background
Prostate cancer (PCa) is one of the most frequently and commonly encountered malignancies among males, especially in the European and American regions [1,2]. In addition, the incidence rate of PCa in Asian regions has signi cantly increased in recent years though advanced therapies including surgery, hormone therapy, chemotherapy and radiation therapy had been applied in PCa patients [3,4]. Besides, it has been reported that PCa is one of the leading causes for cancer-related deaths in men [5,6]. According to the guidelines of European Association of Urology and American Urological Association, the current diagnosis of PCa mainly depends on digital rectal examination (DRE) and increased prostate speci c antigen (PSA), which have several limitations, such as low diagnostic speci city and missed diagnosis [7,8]. What's more, the development of PCa was a slow process and majority of PCa patients are present with no obvious symptoms at early stage [5,9]. Therefore, it is in urgent need to nd novel biomarkers with high sensitivity and speci city to diagnosed PCa at early stage.
MicroRNAs (miRNAs) are a class of short, endogenous, single-stranded, conserved non-coding RNAs that are about 17-25 nucleotides in length in eucaryote cells [10,11]. They can bind to the 3'-untranslated regions (3'-UTR) of a variety of genes to regulate their expression through sequence-speci c base paring at post-transcription level [12,13]. A large number of studies have demonstrate that miRNAs play crucial roles in biological processes, such as cell proliferation, migration, invasion, apoptosis and oncogenesis [14][15][16]. MiR-378, as a member of miRNA family, has also been reported to be involved in cell survival, angiogenesis, and tumor growth [17,18]. Besides, a growing number of investigations have claimed that

Results
Down-regulated expression of serum miR-378 in PCa patients The expression of miR-378 in PCa patients and healthy controls was determined using qRT-PCR. The relative expression level of serum miR-378 in PCa patients was 1.824 ± 0.576, while that in healthy abnormal expression of miR-378 are related with the occurrence of different cancers, such as ovarian cancer [19]. Considerable studies have demonstrated the candidate role of miRNAs in tumor diagnosis and prognosis. However, until now, the diagnostic role of miR-378 in PCa patients was still unclear.
In the present study, we aimed to determine the expression of serum miR-378 in PCa patients and further to assess its diagnostic value for this disease.

Patients and specimens
A total of 117 patients who were pathologically diagnosed as PCa were recruited from Harrison International Peace Hospital. Patients accompanied with other prostatic diseases, such as prostatitis, were excluded from the study. The collected patients received no chemo-or radio-therapy before this investigation. In addition, 88 blood donors in the same hospital were enrolled as healthy controls. An aliquot of 5 ml peripheral blood was collected from all the participants to prepare serum samples. The serum samples were stored in EDTA-containing tubes for later use. This study was conducted with the approval of the Ethics Committee of Harrison International Peace Hospital. The informed consent had obtained from each participant in advance.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from serum samples by Qiagen miRNeasy Mini Kit (Qiagen, GmbH, Germany) following the manufacture's instruction. Then the total RNA was used to synthesize cDNA according the the TaqMan MicroRNA Assay protocol (Applied Biosystems). Finally, the real-time PCR ampli cation was performed using the Applied Biosystems 7599 instrument under optimal conditions. The expression of serum miR-378 was calculated using the 2 −∆∆Ct method. In addition, U6 was adopted as an internal reference.

Statistical analysis
All data in the study were statistically analyzed using Sigmaplot 12.5 and GraphPad Prism 5.0 software.
The student's t-test was used to compare the expression difference of miR-378 between groups. Chisquare test was used to describe the relationship of miR-378 expression and clinical parameters. The receiver operating characteristics (ROC) curve was plotted to calculate the area under the curve (AUC) as well as the sensitivity and speci city of miR-378 in PCa patients. P value was considered to be signi cant if it was less than 0.05. individuals was 3.101 ± 0.875. As shown in Fig. 1, miR-378 was signi cantly decreased in PCa serum compared with healthy controls (P < 0.0001).

Relationship between miR-378 expression and clinical features of PCa patients
To explore the relationship of miR-378 expression with clinical characteristics of PCa patients, the χ 2 test was conducted. Patients were classi ed into high expression group (n = 59) and low expression group (n = 58) according to the median expression level (1.75) of serum miR-378. As listed in Table 1, the increased expression of serum miR-378 was closely related with AR status (P = 0.004), Gleason score (P = 0.013) and TNM stage (P = 0.020). However, no relationship was found between miR-378 expression and age (P = 0.117), serum PSA level (P = 0.298), urine retention (P = 0.117) and NET rate (P = 0.079). PSA: prostate speci c antigen; NED: neuroendocrine differentiation; AR: androgen receptor.

Diagnostic performance of serum miR-378 in PCa
The ROC analysis was conducted to identify the diagnostic role of serum miR-378 in PCa. As shown in Fig. 2, the optimal cutoff point was 1.845, giving the sensitivity of 75.21% and the speci city of 89.77%.

Discussion
PCa is one of the most common male malignant tumors in the world. Due to the large population, aging, or changes of life style, the number of PCa patients has been increased greatly in recent years [20]. Patients at early stage are present with no obvious symptoms, which indicates that there might be local in ltration or distant metastasis when symptoms appear, such as urine retention, di cult urination and hematuria. In recent years, the detection rate of PCa has been signi cantly elevated due to the application of serum PSA and DRE in clinic. Nevertheless, there are several limitations in these typical screening methods. And it is known to all that tumors at early stages have more opportunities to be cured than the advanced ones. Therefore, more and novel speci c biomarkers are needed to early diagnose PCa. In the study of Wu et al., urine PCA3 was used as a diagnostic biomarker for PCa [21]. Casanova-salas et al.
demonstrated that miR-187 and miR-182 has great capacity for diagnosing PCa [22]. In this study, we were engaged in nding more novel biomarkers to better improve the PCa diagnosis.
MiRNAs has been reported to be implicated with plenty of physical and biological processes through regulating the expression of target genes. It is said that miRNAs are abundant in serum, plasma and other body uids with high stability [23]. Besides, miRNAs have similar signatures between men and women with different ages. In the last decade, numerous miRNAs have been investigated as prognostic and diagnostic biomarkers and therapeutic targets. Cao et al. revealed that miR-454 overexpression was correlated with unfavorable prognosis for patients with triple-negative breast cancer [24]. In the study of Wei et al., they reported that miR-1 was signi cantly downregulated in recurrent PCa tissues and it can function as an independent predictive factor for PCa [25]. Moreover, Want et al. showed that miR-378 was related with poor prognosis as well as tumor invasiveness of patients with glioma [26]. In the present study, we attempted to detect the diagnostic role of serum miR-378 in PCa.
Previous studies have demonstrated that serum miR-378 was signi cantly upregulated in several cancers, including gastric cancer, renal cell carcinoma and PCa [23,27,28]. At present, we rst compared the expression levels of serum miR-378 in PCa patients and healthy individuals, and our ndings were highly in accordance with previous results, indicating miR-378 might be related with the progression of PCa. The Chi-square test elucidated that serum miR-378 up-regulation was in uenced by AR status, Gleason score and TNM stage. Based on the above ndings, we further determined the diagnostic role of serum miR-378 in PCa using ROC analysis and the results showed a high AUC value for serum miR-378 in PCa diagnosis, indicating serum miR-378 could distinguish PCa patients from healthy controls.
In this study, we identi ed the diagnostic role of serum miR-378 in PCa for the rst time. However, the precise mechanisms of serum miR-378 on PCa pathogenesis are still not clear. In the study of Qi et al., they found miR-378 was down-regualted in PCa tissues and validated MAPK1 was a direct target of miR-378 in human PCa [29]. Besides, Avgeris et al. also observed the reduction of miR-378 was predicted to target both KLK2 and KLK4 and increases the risk of PCa progression [30]. Therefore, more and further investigations are required for future studies.

Conclusions
In conclusion, we con rmed the down-regulation of serum miR-378 in PCa patients compared with healthy individuals. Positive AR, large Gleason score and advanced TNM stage were associated with miR-378 expression. Furthermore, our results identi ed the potential of serum miR-378 as a promising diagnostic biomarker for PCa. 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.

Figure 1
Expression of serum miR-378 in PCa patients and healthy controls. The result showed that serum miR-378 level was signi cantly lower in PCa patients than that in healthy controls (P<0.0001).