MiR-103 Assumes Signifying Capacity in Prostate Cancer Diagnosis

Background: Prostate cancer is one of the common cancers among males with high incidence and mortality. MiR-103 has been reported to be involved in several human cancers. In our study, we aimed to explore the diagnostic value of miR-103 in PCa. Methods: In this study, qRT-PCR was performed to investigate the expression levels of miR-103 in serum specimens collected from 141 PCa patients and 77 healthy volunteers. MTT and cell invasion assay were performed to detect the effects of miR-103 on the proliferation and invasion of PCa cells. The diagnostic value of miR-103 in PCa was evaluated by ROC curve. Results: The results showed that serum miR-103 levels were higher in PCa patients than that in healthy group (P<0.001). And the expression of miR-103 was signicantly associated with PSA (P=0.007), differentiation (P=0.009), and lymph node metastasis (P=0.037). Cell experiments in vitro demonstrated that miR-103 could promote PCa cell line proliferation and invasion. In addition, ROC analysis suggested that miR-103 could discriminate between PCa patients and healthy individuals at the cut-off value of 0.970. The AUC was 0.885, with the sensitivity of 97.2% and specicity of 81.8%. Conclusions: The increased miR-103 levels can promote proliferation and invasion of PCa cells. Serum miR-103 is signicantly associated with tumor progression, which may be a novel biomarker for PCa diagnosis.


Background
Prostate cancer (PCa) is one of the common malignant tumors in men worldwide [1,2]. Although the cancer is not life threatening for most cases, PCa has become one of the leading causes for cancerrelated death among male patients, especially in the developed countries [3,4]. The low survival rate may be caused by high frequency of metastasis and recurrence [5]. Consequently, early detection is crucially important for PCa prognosis. At the present time, serum prostate-speci c antigen (PSA) is the commonly used screening biomarker for PCa, which improves the early diagnosis of PCa. However, PSA is with low speci city for PCa, leading to over-diagnosis and over-treatment [6]. Therefore, the effective biomarkers with high sensitivity and speci city for early detection of PCa are urgently needed, which may signi cantly improve the survival rate of PCa patients.
MicroRNAs (miRNAs) are a group of small, non-coding RNAs which can inhibit translation or induce target mRNAs decay by binding to the 3'-UTR of their target mRNAs [7,8]. Growing evidences prove that miRNAs take part in various biological processes, including tumorigenesis and diseases development [9,10]. In the previous studies, a variety of miRNAs are con rmed to be related with cancers, such as miR-301a and glioma, miR-21 and breast cancer, miR-503 and colorectal cancer [11][12][13]. MiR-103, one of the cancer-related miRNAs, is a member of the miR-103/107 family which is located on human chromosome 5 [14]. Related studies suggested that abnormal expression of miR-103 was observed in several cancers, including endometrial cancer, colorectal cancer, gastric cancer and so on [14][15][16]. The effects of miR-103 on PCa were also reported in the previous studies. A study carried out by Singh et al. had indicated that high serum levels of miR-103 predicted aggressive progression in patients who received radical prostatectomy [17]. However, the diagnostic signi cance of miR-103 in PCa has been rarely reported in the previous studies, In this study, we aimed to investigate the diagnostic value of miR-103 in PCa patients. Serum level of miR-103 in PCa patients was detected, as well as its association with clinical characteristics. In addition, receiver operating characteristic (ROC) curve was conducted to evaluate the diagnostic value of PCa patients.

Patients and specimens collection
Patients pathologically diagnosed with PCa were recruited in the study which was carried out in Zhongnan Hospital of Wuhan University. None of the patients received any chemotherapy or radiotherapy before specimens collection. In addition, healthy volunteers were collected as healthy control. Among the healthy group, no one had been diagnosed with any malignancy. 5mL blood specimens were collected from all the participants on the morning after fasting for 8-10h. The blood specimens were stored in tubes with EDTA and then centrifuged for 10min at 2500rpm. The up-stream was serum sample which was stored at -80℃ until used.
Our study was approved by the ethics committee of Zhongnan Hospital of Wuhan University, and the written consents were obtained from all the participants before sampling. The clinicopathological features of the patients were recorded at the beginning of the study, including age, tumor size, PSA, differentiation, TNM stage, and lymph node metastasis.

Cell culture and transfection
Human PCa cell line DU145 was purchased from American Type Culture Collection (ATCC, Manassas, VA). Then the DU145 cells were cultured in RPMI-1640 medium added with 100 IU/ml penicillin, 100 mg/ml streptomycin, 20mM glutamine and 10% heat-inactivated FBS at 37℃ in a 5% CO 2 incubator.
MiR-103 mimics, miR-103 inhibitor and its negative control were purchased from Genepharma Co., Ltd. (Shanghai, China). DU145 cells were transfected with miR-103 mimic, miR-103 inhibitor or NC using Lipofectamine 2000 (Invitrogen, Life Technologies, CA) following the manufacturer's instructions. After transfection, qRT-PCR was applied to detect the relative expression of miR-103 in the cells, to evaluate the effects of transfection. Then cells were harvested for proliferation and invasion analyses after cultured 48 h.

MTT assay
In order to investigate the effects of miR-103 on DU145 cells proliferation, we performed MTT assay. After transfection, 200μl cell suspension were added into 96-well plates (10 2 -10 3 cells/well) for continual 3-5 days in a 5% CO 2 incubator at 37°C. MTT assay was performed every 24 h. 20μl MTT (Sigma-Aldrich, 0.5 mg/ml) was added to each well, and incubated for 1h continuely, and then resolved with 150μl DMSO (Sigma-Aldrich) in each well following low speed oscillation for 10 min. The absorbance values were measured at 570 nm with a spectrophotometer (Bio-Rad Laboratories, Hercules, CA). Every experiment was performed in triplicate.

Cell invasion assay
To measure cell invasion, transwell assay with matrigel was performed. Migration was diluted with serum-free medium (1:3), and then added 25μl migration into the upper well of the Transwell for 30min at 37°C. After digested, the transfected cells were resuspended with serum-free medium. 100μl 5×10 4 transfected DU145 cells were seeded into Transwell chambers (Corning Inc., Corning, NY) and and 500μl FBS were added into the lower chamber. Then the assay plates were incubated at 37°C, 5% CO 2 . After 24 h, discard the medium, washed twice with FBS and then xed with methanal for 30 min. The cells which did not invasive were erased with swab after stained with 0.1% crystal violet for 1 min. The cell number was counted under a microscope. Every experiment was select at least three visions.

Statistical analysis
In this study, all statistical analyses were performed with software of SPSS 19.0 and GraphPad Prism 5. The miR-103 expression level was expressed as mean ± SD and analyzed by student's t tests. The association between serum miR-103 expression and various clinicopathological characteristics were assessed using Chi-square tests. To determine the diagnostic performance of serum miR-103 expression in PCa, the ROC curve was conducted. P values less than 0.05 were considered statistically signi cant.

Demographic characteristics of the study subjects
In the present study, 141 PCa patients and 77 healthy individuals were collected in. The average age of the test group was 60.25 ± 21.03 years, which was similar with the control group. The clinical characteristics of the PCa patients were listed in Table 1.

Higher expression of miR-103 in PCa patients
Serum levels of miR-103 expression in PCa patients and healthy individuals were detected by qRT-PCR.. The results showed that miR-103 expression was signi cantly higher in PCa patients than that in healthy volunteers (P<0.001) (Figure 1).

Association between miR-103 expression and clinical features in PCa patients
In this study, chi-square test was used to investigate the association between miR-103 expression level and the clinicopathological data of PCa patients. The results suggested that miR-103 expression was markedly associated with PSA (P=0.007), differentiation (P=0.009), and lymph node metastasis (P=0.037). However, there was no signi cant association between miR-103 levels and other clinical characteristics, such as age, tumor size, or TNM stage (P>0.05 for all) ( Table 1).

Effects of miR-103 on PCa cells
To investigate the effects of miR-103 on PCa cells, the transfected cells respectively speci c to miR-103 mimic, miR-103 inhibitor and NC were conducted. MiR-103 levels of transfected cells were detected and the results suggested that compared with NC cells, miR-103 levels were markedly increased in miR-103 mimic cells, while the levels were signi cantly decreased in miR-103 inhibitor cell (P<0.05 for both) (Figure 2a). The results revealed that we successfully conducted transfected cells.
MTT assay was carried out to evaluate the effects of miR-103 on DU145 cells proliferation. The results indicated that after transfection for 48h, the growth rate were signi cantly increased in miR-103 mimic transfected cells than that in NC group, while the proliferation of miR-103 inhibitor group was markedly inhibited (P<0.05 for all) (Figure 2b).
In addition, invasion analysis suggested that compared with NC cells, knock-down of miR-103 expression could inhibit DU145 cells invasion. Moreover, over-expression of miR-103 could promote cell invasion (P<0.05 for both) (Figure 2c).

Diagnostic value of miR-103 expression in PCa
ROC curve was conducted to evaluate the diagnostic signi cance of miR-103 in PCa patients. Analysis results indicated that miR-103 could distinguish the PCa patients from the healthy individuals with the sensitivity of 87.2% and the speci city of 81.8%. The cut-off value was 0.970, and the AUC was 0.885 ( Figure 3).

Discussion
PCa is a frequently diagnosed male malignant tumor [18]. Several factors may contribute to its high morbidity, including age, family history, race, and genetic alterations [19]. Although the etiology of PCa remains unknown, a variety of molecules are con rmed to be related with indication and development of PCa, which can used for diagnostic and prognostic biomarkers for the cancer. A study carried out by Cai et al. demonstrated that dual-speci city phosphatase 5 (DUSP5) negatively regulated PCa progression, which could be a prognostic marker for the cancer [20]. A related study scheduled in in vitro suggested that knock-down of NIMA-related kinase 2 (NEK2) could inhibit PCa cell proliferation, indicating NEK2 was signi cantly associated with development of PCa [21]. All of the related studies revealed that genetic factors play important roles in PCa progression and their abnormal expression may serve as non-invasive biomarkers.
MiRNAs are highly conserved in species and act as important roles in various physiological and pathological processes, including developmental abnormalities, autoimmune diseases and cancers [22][23][24]. In the present study, we investigated the diagnostic value of miR-103 in PCa. QRT-PCR was applied to detect the serum levels of miR-103 in PCa patients and healthy individuals. Analysis results indicated that miR-103 was up-regulated in PCa patients, compared with healthy controls. Moreover, the elevated levels were signi cantly associated with high PSA levels, poor differentiation, and positive lymph node metastasis. The results might suggest that miR-103 was an oncogene in PCa, which could promote malignant development of the cancer. However, the mechanisms for miR-103 regulating PCa were not investigated in the study, which was needed to be identi ed in the next study.
In the present study, cell experiments were carried out to evaluate the effects of miR-103 on PCa cell lines in vitro. Our results showed that over-expression of miR-103 could promote PCa cell proliferation and invasion, while knock-down of its expression would inhibit PCa cell growth. However, a related study carried out by Fu et al. held the different opinions [25]. In their study, miR-103 was proved to be a tumor suppressor gene, which could inhibit PCa cell proliferation, invasion and promote apoptosis. Several factors might contribute to the differences, such as the cell types, detection tools, and analysis methods. Therefore, lots of studies were needed to investigate the effects of miR-103 on different PCa cell lines based on the same experiment condition.
In the previous studies, several miRNAs were con rmed to serve as biomarkers for PCa. Wang et al. had reported that serum miR-410-5p level was signi cantly higher in PCa patients than that in the healthy controls and the further analysis suggested that miR-410-5p could serve as a diagnostic marker for PCa [26]. A related meta-analysis demonstrated that down-regulated miR-145 predicted poor outcomes for patients diagnosed with PCa [27]. The study carried out by Sun et al. suggested that the decreased expression levels of miR-128 in tissues and serum specimens of PCa patients were signi cantly associated with aggressive tumor progression, which might serve as a marker for PCa diagnosis and prognosis [28]. All of the studies suggested that various miRNAs were involved in the initiation and development of PCa, which could act as biomarkers for the cancer. In the current study, we investigated the diagnostic value of miR-103 in PCa. Analysis results suggested that miR-103 could distinguish the PCa patients from the healthy control with high sensitivity and speci city. However, the signi cance of miR-103 used for PCa diagnosis in clinical practices was needed to be investigated in the further study with large sample size.

Conclusions
In conclusion, the elevated levels of miR-103 can promote PCa cell proliferation and invasion, which may be signi cantly correlated malignant tumor progression. Moreover, serum miR-103 may be a potential biomarker for PCa diagnosis. 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.

Competing interests
The authors declare that they have no competing interests.

Funding
Not applicable.
Authors' contributions S.L., T.L. and X.R. conceived and designed the experiments; S.L., T.L. and X.R. conceived and performed the experiments; S.L., T.L. and X.R. prepared gures. S.L., T.L. and X.R. wrote the main manuscript text. All authors reviewed the manuscript.  Figure 1 Serum miR-103 expression levels evaluated by qRT-PCR in PCa patients and healthy controls. Analysis results indicated that serum miR-103 levels were signi cantly higher in PCa patients than that in the healthy controls. ***: suggested P<0.001.