Downregulation of Urinary microRNA-200c acts as a Promising Novel Bio-marker in the Diagnosis of Patients With Bladder Cancer

(miRNAs) in various to explore the diagnostic performance of Quantitative real-time polymerase chain reaction (qRT-PCR) method was applied to measure the relative expression of urine miR-200c in bladder cancer patients. The relationship between urine miR-200c level and clinicopathological factors was analyzed using χ 2 test. The diagnostic capacity of urine miR-200c was calculated using the receiver operating characteristics (ROC) curve analysis.


Background
Bladder cancer is one of most common malignancy in urinary system around the world [1]. It is featured by high recurrence, leading to poor outcomes and high mortality [2]. Currently, the standard of diagnostic tool for urinary bladder cancer is cystoscopy, but its clinical values are limited due to the invasive procedure, high cost and low e cacy in at tumors like carcinoma in situ [3]. Non-invasive urine cytology, a convenient test, is used as an adjunct to cystoscopy [4]. It has high diagnostic speci city, but its sensitivity is unsatisfactory, especially for low-grade tumor [5]. Recently, there are numerous urine-based tests for early screening of bladder cancer, such as bladder tumor antigen (BTA), nuclear matrix protein 22 (NMP22) and FISH [6,7]. However, none of them have been used in clinical guidelines [8]. Thus, it is of great value to identify non-invasive diagnostic markers for the detection of bladder cancer.
MicroRNAs (miRNAs) are small non-coding RNA molecules that play important roles in various biological processes through modulating the activity of speci c mRNA [9]. Circulating miRNAs stably exist in a variety of body uids including serum, plasma, saliva, pleural uid and urine, and their abnormal expression may lead to human diseases, like cancer [10,11]. Increasing evidences have showed that miRNAs are implicated in human carcinogenesis by functioning as either oncogenes or tumor suppressors [12,13]. The expression patterns of miRNAs show close association with tumor development and progression, which could be employed as predictive biomarkers and therapeutic targets [14]. , belonging to miR-200 family, is located on on chromosome 12p13 [15]. MiR-200c is a well-known tumor suppressor, and its downregulation has been observed in various cancers, such as breast cancer, gastric cancer, etc [16,17]. In bladder cancer, it has been reported that the ectopic expression of miR-200c could inhibit bladder cancer cell proliferation and invasion in vitro [18]. MiR-200c may be involved in tumorigenesis of bladder cancer, which may be employed as an indicator for early screening of the malignant disease. However, there are few reports to address the issue.
In the present study, we examined the relative expression of urine miR-200c in bladder cancer patients, and analyzed the correlation of urine miR-200c expression with clinicopathological parameters of bladder cancer patients. We also investigated the diagnostic performance of urine miR-200c in bladder cancer patients.

Patients and urine samples
The study was approved by the Ethic Committee of Huaihe Hospital of Henan University. All the participants had signed the written informed consents.  Table 1. In addition, 96 age-and gendermatched healthy volunteers were recruited from the physical examination center of the hospital. All the healthy controls had no evidences for urological disorders or malignant diseases. From each subject preoperative urine sample (50ml) was collected in a sterile container and immediately refrigerated at the urology clinic. Within 2 hours urine samples were transported to the laboratory on ice. Each urine sample was assigned a unique identifying number before immediate laboratory processing.
The sample was centrifuged at 3000g at 4℃ for 10 minutes. The urine supernatant aliquots was decanted, centrifuged at 12000g for 10 minutes at 4℃ and stored at -80℃ before analysis at a genitourinary tissue bank.
RNA extraction and quantitative real-time transcriptase polymerase chain reaction (qRT-PCR) Total RNA was extracted from urine samples using mirVana miRNA isolation kit (Ambion, Austin, TX, USA), according to the manufacturer's directions. The concentration and purity of the RNA samples were measured using spectrophoto, and the RNA samples with the OD260/280 value of 1.8-2.0 were used for the subsequent analysis. First strand cDNA was synthesized from the total RNA using a universal cDNA synthesis (Exiqon, Vedbaek, Denmark) in accordance with the manufacturer's instructions. The relative expression of miR-200c was estimated using qRT-PCR method which was performed with SYBR Prime Script miRNA RT-PCR kit (Takara, Japan) in ABI 7500 real-time PCR system (Applied Biosystems, USA). RNU6B was used as the reference control. All of the samples were tested in triplicate. The relative level of miR-200c was calculated using the 2 -ΔΔCt method.

Statistical analysis
All the statistical analyses were performed using SPSS 18.0 software (SPSS software, Inc, IL, USA), and graphs were plotted using Origin 9.0. The Student's t test was performed to compare urine miR-200c levels between bladder cancer patients and healthy controls. The relationship between urine miR-200c level and clinicopatholoigcal features was analyzed by χ 2 test. Receiver operating characteristics (ROC) curves were established to evaluate the diagnostic performance of miR-200c in distinguishing bladder cancer patients from the controls. All statistical tests were two-sided and P values less than 0.05 were considered as statistical signi cance.

Results
The relative expression of urine miR-200c in bladder cancer patients The expression level of miR-200c was detected using qRT-PCR in 205 urine samples (109 bladder patients and 96 controls). As shown in Figure 1, compared to healthy controls, urine miR-200c expression was signi cantly decreased in bladder cancer patients (P=0.000).

The correlation between urine miR-200c expression and clinicopathological characteristics
To identify the potential association between urine miR-200c expression and clinicopatholoigcal parameters in bladder cancer, the patients were divided into two groups based on the median value  The diagnostic e cacy of urine miR-200c in bladder cancer patients ROC curve analyses revealed that urine miR-200c could use as a novel non-invasive biomarker to discriminate bladder cancer patients from healthy controls with an AUC value of 0.844 (95%CI: 0.789-0.898) (Figure 2). At a cutoff value of 1.235, the optimal sensitivity and speci city were 89.0% and 70.7% respectively.

Discussion
Bladder cancer is a heterogeneous disease with unpredictable clinical outcome. Several risk factors have been identi ed for bladder cancer, such as cigarette smoking, exposures to aromatic amines and 4,4'methylenebis(2-chloroaniline) [19]. However, the molecular mechanisms underlying the bladder cancer development are still unclear. Nowadays, early screening and detection of bladder cancer remain a great challenge in clinic. Therefore, it is important to explore the effective bio-markers for early diagnosis of bladder cancer.
MiRNAs are a group of small endogenous RNAs, and they play regulatory role in gene expression at posttranscriptional level [9]. The expression pro le of extracellular cell-free miRNAs are signi cantly correlated with tumor initiation, development and progression, which have been considered as promising biomarkers for cancer management [20]. The urinary miRNAs are stable at room temperature, and they could not be unaffected by multiple freeze-thaw cycles [21], indicating their properties as molecular biomarkers for cancer diagnosis and prognosis. Urine supernatant is a particularly desirable source of biomarkers for bladder cancer, with greatly convenient, reducing protein interference during RNA extraction and re ecting the status of diseases [22].
MiR-200c belongs to the miR-200 family which consists of miR-200a, miR-200b, miR-200c, miR-141 and miR-429 [23]. Accumulating evidences have demonstrated that miR-200 family has the capacity to regulate cancer transformation, growth, metastasis, and therapeutic response through targeting multiple signaling pathways, such as epithelial-mesenchymal transition (EMT), TGF-β signaling, PI3K/Akt signaling, Notch signaling, VEGF signaling, and NF-κB signaling [15,24]. The expression pattern of miR-200c has been con rmed as a potential diagnostic and prognostic biomarker in a variety of tumors. For instance, Antolín et al. reported that circulating miR-200c was deregulated in breast cancer and showed close correlation with clinical characteristics of the patients that might serve as an independent prognostic indicator for the patients [25]. Meng et al. revealed that miR-200c level was involved in tumor progression and could distinguish malignant cases from benign ovarian tumors [26]. However, the clinical signi cance of miR-200c had been rarely reported in bladder cancer.
In the present study, qRT-PCR was applied to detect the relative expression level of miR-200c in 205 urine samples collected from 109 bladder cancer patients and 96 healthy controls. Urine miR-200c expression was obviously down-regulated in bladder cancer patients compared with healthy controls. Additionally, the decreased expression of miR-200c was negatively correlated with histological grade, tumor stage, and lymph node metastasis. All the data suggested that miR-200c as a tumor suppressor was involved in the progression and metastasis of bladder cancer patients. ROC analysis illustrated that urine miR-200c was an useful biomarker for early detection of bladder cancer. The conclusion was consistent with the previous studies. Wu et al. reported that the urinary miR-200 family levels were decreased in patients with bladder cancer [27]. The study carried out by Liu et al. showed that miR-200c could inhibit bladder cancer cells proliferation through down-regulating BMI-1 and E2F3 [28]. However, the possible underlying mechanisms for its participation in bladder cancer progression remained not clarify. Additionally, the sample size was relatively small that might in uence the accuracy of our results. Therefore, further researches with a extended sample size will be performed to improve our conclusion.

Conclusion
In summary, urine miR-200c expression is signi cantly decreased in bladder cancer patients, and negatively correlated with malignant disease progression. Urine miR-200c may be a potential noninvasion biomarker for early diagnosis of bladder cancer. The authors report no con icts of interest in this work. Ethics approval and consent to participate This study was supported by the Ethics Committee of Huaihe Hospital of Henan 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. Data availability Data sharing is not applicable to this article as no datasets were generated or analysed during the current study. Funding No funding was received Competing interests The authors declare that they have no competing interests.
Authors' contributions H.Z. design of the work; WL.T. the acquisition, analysis, L.G. interpretation of data; ZH.Y. the creation of new software used in the work; XD.B., XD.L. have drafted the work or substantively revised it. All authors read and approved the nal manuscript.