Urine MACC1 Plays an Indicative Role in Diagnosing Bladder Cancer

Background: Present study aimed to estimate the diagnostic ecacy of urine Metastasis-associated in colon cancer 1 (MACC1) for bladder cancer patients. Methods: Urine MACC1 expression was measured in bladder cancer patients and controls via quantitative real-time polymerase chain reaction (qRT-PCR) assay. Chi-square test was used to assess the correlation of urine MACC1 levels with clinicopathological features of the patients. The diagnostic performance of urine MACC1 was evaluated by receiver operating characteristics (ROC) curve. Results: Urine MACC1 was obviously elevated in bladder cancer patients compared with controls (P<0.001). What’s more, abnormal urine MACC1 expression was signicantly correlated with differentiation (P=0.013), tumor stage (P=0.023), lymph node metastasis (P=0.033) and distant metastasis (P=0.026). Furthermore, according to ROC analysis, urine MACC1 could distinguish bladder cancer patients from controls with an AUC (the areas under the ROC curve) of 0.909. The cut-off value of urine MACC1 for bladder cancer diagnosis was 1.275, with 79.8% specicity and 87.3% sensitivity. Conclusion: Urine MACC1 may be act as a biomarkers for diagnosis of bladder cancer.


Background
Bladder cancer is one of the most frequently diagnosed malignancies in the urinary system around the world [1]. About 70-80% cases are con rmed as non-muscle-invasive (Ta and T1 tumors) bladder cancer (NMIBC), and the rest belongs to be muscle-invasive (T2-T4 tumors) bladder cancer (MIBC) [2]. Although the NMIBC is not life-threatening, with a ve year-survival more than 90% [3]. However, approximate 50-70% of NMIBC will relapse, and 10-15% of them progress to MIBC within ve-year of diagnosis [4]. Therefore, early diagnosis and postoperative monitoring are crucial for treatment and clinical outcomes of bladder cancer patients. At present, cystoscopy is the golden standard for early screening and monitoring of bladder cancer. However, the high cost and invasive procedures may limit its wide application [5]. Therefore, non-invasive biomarkers are in urgent need for patients with bladder cancer.
The genetic materials, such as protein, DNA and RNA, derived from bladder tissues could be detected by easy methods in urine specimens that are considered as promising candidates for early detection and monitoring of bladder cancer [6,7].
Metastasis-associated in colon cancer 1 (MACC1) initially detected in colon cancer [8] is con rmed a new cancer-related gene, located on human chromosome 7 (7p21.1). MACC1 has the capacity to induce cell proliferation, migration and invasion [9,10]. Moreover, numerous reports have proved that MACC1 was highly expressed in human tumors, such as nasopharyngeal carcinoma, glioma, gastric cancer and so on [11][12][13]. In bladder cancer, Xu et al. reported that knockdown of MACC1 using RNA interference could induce inhibition on proliferation and invasion of bladder cancer cells in vitro [14]. However, the expression patterns of urine MACC1, as well as its diagnostic e cacy for bladder cancer remained poorly known.
In the present study, we measured the expression status of MACC1 in urine samples collected from bladder cancer patients and non-malignant controls. Moreover, the relationship between urine MACC1 level and clinicopathological factors was also analyzed. In addition, the diagnostic value of urine MACC1 was evaluated in bladder cancer patient.

Patients and sample collection
The study was examined and approved by the Ethical Committee of Huaihe Hospital of Henan University. All participants signed written informed consents in the study.
118 patients who were newly diagnosed with bladder cancer were recruited from Huaihe Hospital of Henan University. Diagnosis of bladder cancer was based on pathological examinations. None of the patients had received any anti-tumor treatments. The demographic data and medical history of the patients were recorded in Table 1. Tumor classi cation was established according to Union for International Cancer Control (UICC) 2010 TNM system. At the same time, 104 age-and gender-matched subjects, including 92 healthy volunteers and 12 subjects with urolithiasis, were collected from the same hospital as controls. Urine samples were collected from each participant in the morning, and then centrifuged at 600 × gravity for 5 minutes at 4℃. Supernatant was decanted, aliquoted into Eppendorf tubes and maintained at -80℃ until use.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from urine samples using TRIzol reagent (Invitrogen, Grand Island, NY, USA) based on the protocols of the manufacturer. The concentration and purity of RNA were measured at 260 and 280 nm with BioPhotometer plus (Eppendorf AG, Hamburger, Germany).
Then the rst chain of cDNA was generated by reverse transcription utilizing the HiFi-MMLV cDNA Kit (Beijing CoWin Biotech Co. Ltd., Beijing, China) according to the instruction of the manufacturer. QRT-PCR was carried out using a SYBR green I Master Mix kit (Invitrogen). The primers sequence was synthesized according to the previous report [14]. β-actin was employed as reference control. The expression of MACC1 gene was normalized to the internal control, and calculated using 2 −ΔΔCt method. Each test was repeated at least three times.

Statistical analysis
All statistical analyses were carried out using SPSS version 18.0 (SPSS, Chicago, USA), and graphs were plotted by GraphPad Prism 5.0 (GraphPad Software). The statistical differences of MACC1 expression between bladder cancer patients and controls were assessed via independent Student t test. Chi-square test was applied to analyze the association of urine MACC1 with clinicopathological features of bladder cancer patients. The diagnostic capacity of urine MACC1 for bladder cancer was evaluated by receiver operating characteristics (ROC) curve. The speci city and sensitivity, as well as the area under the curve (AUC) value were calculated to estimate the diagnostic performance of urine MACC1 for bladder cancer. P value less than 0.05 was regarded as statistical signi cance.

Results
Baseline characteristics of the bladder cancer patients

Urine MACC1 level in bladder cancer patients
We analyzed the expression levels of MACC1 mRNA in urine samples of 118 bladder cancer patients and 104 controls. According to qRT-PCR assay, urine MACC1 levels were dramatically up-regulated in bladder cancer patients, compared to the controls (P < 0.001, Fig. 1).

Association of urine MACC1 with clinicopathological parameters
To further analyze the relationship between urine MACC1 levels and clinicopathological parameters, we divided all patients into two groups based on the median value of urine MACC1 expression: low MACC1 expression group (n = 54) and high MACC1 expression group (n = 64). As indicated in Table 1, the elevated urine MACC1 expression was distinctly correlated with bladder cancer patients' differentiation (P = 0.013), tumor stage (P = 0.023), lymph node metastasis (P = 0.033), and distant metastasis (P = 0.026).
Nevertheless, urine MACC1 levels had no association with other clinicopathological factors, such as age, gender, or tumor size (all P > 0.05).
Diagnostic performance of urine MACC1 in bladder cancer ROC curve analysis illustrated that urine MACC1 could distinguish bladder cancer patients from healthy controls with an AUC (the areas under the ROC curve) of 0.909 (95%CI: 0.872-946) (Fig. 2). At a cutoff value of 1.275, the optimal speci city and sensitivity values were 79.8%, and 87.3%, respectively (Fig. 2).

Discussion
Bladder cancer is a heterogeneous disease with unpredictable clinical course, representing a leading cancer-related deaths [15]. At present, there are several major risk factors identi ed in bladder cancer, such as cigarette smoking, chronic infection by Schistosoma haematobium, and exposure to the carcinogens [16,17]. Early diagnosis and close monitoring are key for clinical outcomes of patients with bladder cancer. Unfortunately, there are no recommended biomarkers for bladder cancer in routine clinical application until now [18]. In current study, we con rmed that the expression pro les of urine MACC1 were signi cantly different between bladder cancer patients and non-malignant individuals. Urine MACC1 might be a potential diagnostic biomarker for bladder cancer.
Growing evidences have demonstrated that the over-expression of MACC1 may contribute to malignant tumor progression through enhancing cell proliferation, migration and invasion [8,[20][21][22]. The expression pattern of MACC1 in human uids may have the possible to serve as a biomarker for early diagnosis and prognosis evaluation in several types of human cancers. For instance, Ashkorab et al. reported that MACC1 transcripts were obviously increased in plasma samples of colon adenoma patients compared with normal patients. Moreover, plasma MACC1 could distinguish the colon adenoma patients from healthy individuals [23]. Tan et al. showed that serum MACC1 was highly expressed in breast cancer patients, and it might be a reliable biomarker for early diagnosis and prognosis evaluation in breast cancer patients [24]. A study carried out by Wang et al., reported that plasma MACC1 expression exhibited signi cantly increased in non-small cell lung cancer patients, and might be a promising non-invasive factor for diagnosis and prognosis of the disease [25]. Taken together, the expression of MACC1 in human uids shows speci c to cancer, which may be employed as biomarker for diagnosis and monitoring of cancer. However, the clinical signi cance of urine MACC1 in bladder cancer was rarely reported.
In this study, urine MACC1 mRNA expression was examined in bladder cancer patients and controls using qRT-PCR method. The expression of MACC1 was signi cantly increased in urine samples of bladder cancer patients in comparison to controls. The result was in line with the previous study reported by Xu et al., revealing that MACC1 mRNA levels were up-regulated in human urothelial carcinoma [14]. In addition, we estimated the association of urine MACC1 expression with clinicopathological parameters of bladder cancer patients. The abnormal MACC1 expression in urine samples was remarkably associated with tumor differentiation, tumor stage, lymph node metastasis, and distant metastasis. The bladder cancer patients with high expression of MACC1 were more likely to undergo malignant disease progression. The in vitro experiments carried out by Xu et al. suggested that MACC1 could regulate the biological behaviors of human bladder urothelial carcinoma cell line T24 [14]. Over-expression of MACC1 might contribute to progression of bladder cancer through enhancing the malignant biological behaviors of cancer cells.
Given the different levels of urine MACC1 in bladder cancer cases and healthy individuals, we hypothesized that urine MACC1 might be a potential diagnostic biomarker for bladder cancer. ROC curves were plotted to estimate the diagnostic performance of urine MACC1 in bladder cancer. Urine MACC1 could distinguish bladder cancer patients from the non-malignant controls with high sensitivity and speci city. Urine MACC1 might act as a valid and reliable biomarker for early screening of patients suffering from bladder cancer.
This study had certain limitations. Firstly, the number of patients recruited in the study was relatively small, and further studies with a large scale number of patients are required to con rm our ndings. Secondly, the patients corrected in our study all came from one hospital that might cause bias to our results. In addition, the molecular mechanisms underlying the oncogenic function of MACC1 remained poorly known. Further in vitro and in vivo experiments are required to address the issues. Therefore, multicenteric studies should be considered to verify the value of MACC1 as a diagnostic biomarker for bladder cancer.

Conclusions
In conclusion, urine MACC1 level is up-regulated in bladder cancer patients and closely associated with tumor metastasis and progression. Urine MACC1 may be a valuable biomarker for early screening of bladder cancer.

List Of Abbreviations
Metastasis-associated in colon cancer 1 (MACC1) Quantitative real-time polymerase chain reaction (qRT-PCR) 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
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.  Expression of urine MACC1 determined by qRT-PCR assay in bladder cancer patients and controls. Urine MACC1 levels were signi cantly increased in bladder cancer patients compared with controls (***: P<0.001).

Figure 1
Expression of urine MACC1 determined by qRT-PCR assay in bladder cancer patients and controls. Urine MACC1 levels were signi cantly increased in bladder cancer patients compared with controls (***: P<0.001).
Page 13/14 Receiver operating characteristics (ROC) curve analysis for the diagnostic accuracy of urinary MACC1 in bladder cancer. The AUC (areas under the ROC curve) value was 0.909 (95%CI: 0.872-0.946). The speci city and sensitivity were 79.8% and 87.3%, respectively. All the data revealed that urine MACC1 could distinguish bladder cancer patients from the controls, and the cut-off value was 1.275.