LncRNA CASC2 Plays an Indicative Role in Diagnosing Bladder Cancer


 Background: Bladder cancer is a common cancer of urinary system, with high incidence and mortality. LncRNA CASC2 as a tumor suppressor has been reported to be involved in many human tumors. In this study, we aimed to explore the diagnostic value of CASC2 for bladder cancer patients.Methods: qRT-PCR was used to detect the expression level of CASC2 in 140 bladder cancer patients and 90 healthy volunteers. The differences of CASC2 expression between the cancer group and healthy group were analyzed using student’s t test. The correlation of CASC2 expression with clinical characteristics of the bladder cancer patients was estimated with Chi-square test. In addition, ROC curve was plotted to evaluate the diagnostic value of CASC2 for bladder cancer patients.Results: Serum CASC2 level was lower in bladder cancer patients than that in healthy group (P<0.05). The expression level of CASC2 was significantly associated with histological grade (P=0.000), TNM stage (P=0.000), and lymph node metastasis (P=0.001). The area under the ROC curve (AUC) was 0.864 and the optimal cutoff value was 0.955, suggesting the diagnostic value of CASC2 for bladder cancer. The diagnostic sensitivity was 77.8% and specificity was 85.7%.Conclusion: CASC2 may be a novel biomarker for early diagnosis of bladder cancer.


Background
Bladder cancer is a most common cancer in urogenital system around the world [1,2]. Moreover, its morbidity is signi cantly higher in males than that in females [3]. A variety of risk factors have been con rmed for bladder cancer, including tobacco smoking, exposure to certain chemicals in the working, as well as genetic factors [4]. At present, the treatments for bladder cancer have been signi cantly improved [5], but the prognosis of the patients are not obviously improved, due to the delay in early diagnosis [6,7]. In bladder cancer, the 5-year survival rate of the patients with early stages is more than 75%, but the 5-year survival rate of patients with advanced stages is very low [8]. Thus, it is crucial to explore effective biomarkers to achieve early diagnosis of bladder cancer.
Long non-coding RNAs (LncRNAs) are group of non-coding RNAs, with the length of more than 200 nucleotides [9]. Despite of limited protein coding ability, lncRNAs can regulate the expression of genes at both the transcriptional and post-transcriptional levels [10]. LncRNAs are involved in various physiological processes, such as cell apoptosis, proliferation, differentiation, and the dysregulation of lncRNAs may lead to human diseases, especially cancer [11]. The abnormal expression expression of lncRNAs is observed in many human tumors, such as esophageal squamous cell carcinoma, thyroid carcinoma, etc [12,13]. Cancer susceptibility candidate 2 (CASC2) is is located in 10q26 chromosome, belonging to lncRNA family [14]. LncRNA CASC2 was rstly con rmed as a tumor suppressor in endometrial cancer [15]. Then, the abnormal expression of CASC2 has been observed in a number of cancers [16]. In bladder cancer, it was reported that the expression of CASC2 was down-regulated, and could affect the biological behaviors of bladder cancer cells [14]. However, the diagnostic performance of CASC2 expression for bladder cancer patients had been rarely reported in the published articles.
In this study, we aimed to detect the serum level of CASC2 in bladder cancer patients and its functional roles in caner progression. In addition, we investigated the diagnostic performance of serum CASC2 for bladder cancer.

Study subjects
A total of 140 bladder cancer patients were recruited from Huaihe Hospital of Henan University, and they were all diagnosed by pathologists. 90 healthy volunteers were recruited as the control group. In the healthy group, no one had been diagnosed with any malignancies. This study was approved by the ethics committee of Huaihe Hospital of Henan University. The written consents were obtained from all the subjects. The clinical features of the patients were listed in Table 1, including age, gender, tumor number, tumor size, histological grade, TNM stage, and lymph node metastasis. After a 12 h-fasting, 5 mL peripheral blood was collected from all the bladder cancer patients and healthy volunteers using a blood collection tube with EDTA. Then serum specimens were isolated from blood through centrifugation, and stored at -80℃ until used.

Statistical analysis
In this study, all statistical analyses were performed with software of SPSS 19.0 (SPSS Inc., Chicago, IL) and GraphPad Prism 5 (GraphPad Software, Inc., USA). The expression of CASC2 was expressed as mean ± SD, and student's T test was used to analyze the differences of the CASC2 expression between the tumor and healthy samples. The bladder cancer patients were divided into two groups according to the average expression of CASC2. The relationship between serum CASC2 expression and various clinical characteristics of bladder cancer patients was assessed with Chi-square tests. To determine the diagnostic value of serum CASC2 in bladder cancer patients, receiver operating characteristic (ROC) analysis was performed. P values < 0.05 were considered statistically signi cant.

Results
The expression level of CASC2 in bladder cancer tissues QRT-PCR was used to detect the expression level of CASC2 in bladder cancer patients and healthy volunteers. As showed in Fig. 1, the expression of CASC2 was signi cantly lower in bladder cancer patients than that in healthy group (P < 0.05).
The association of CASC2 expression with the clinical features of bladder cancer patients Chi-square test was used to detect the correlation between CASC2 expression level and the clinical data of bladder cancer patients. The results showed that CASC2 expression was signi cantly associated with histological grade (P = 0.000), TNM stage (P = 0.000), and lymph node metastasis (P = 0.001). Meanwhile, CASC2 expression had no signi cant correlation with the age, tumor number or tumor size (all P > 0.05) ( Table 1).
The diagnostic value of CASC2 in bladder cancer patients ROC analysis was performed to estimate the diagnostic value of CASC2 for patients with bladder cancer.
The results were showed in Fig. 2. From the results, we could see that CASC2 could distinguish bladder cancer patients from the healthy volunteers with the area under the curve (AUC) value of 0.864. The diagnostic sensitivity was 77.8%, and the speci city of 85.7%. The cutoff value of CASC2 expression for bladder cancer diagnosis was 0.955.

Discussion
Bladder cancer is a fatal disease, with high metastasis ability [17]. There are various available treatments for bladder cancer patients, including radiation therapy, immunotherapy, radical cystectomy, and postoperative instillation of chemotherapy [18,19]. However, the clinical outcomes of the patients have not been signi cantly improved. The prognosis of the patients is closely correlated with the tumor stages.
Early diagnosis is very important for the prognosis of bladder cancer patients. Currently, the early detection of bladder cancer mainly dependents on cystoscopy and voided urinary cytology. However, the clinical values are limited due to invasive and uncomfortable procedures, low sensitivity and high cost [20,21]. Therefore, the novel molecular biomarkers are in urgent need for early diagnosis of bladder cancer. In this study, we aimed to explore a novel biomarker to achieve the early diagnosis of bladder cancer.
CASC2 is a novel lncRNA in human genome, and it was rstly discovered by Baldinu, P et al. in human endometrial cancer [15]. The abnormal expression of CASC2 is found in many human cancers, and it can act as a tumor suppressor [22]. For instance, Wang et al. found that in glioma tissues and cell lines the expression level of CASC2 was down-regulated [23]. The study of He et al. found the expression of CASC2 was lower in non-small cell lung cancer (NSCLC) tissues and was correlated with tumor size and tumor stage [24]. The study group of Cao et al. indicated that CASC2 expression was down-regulated in human renal cell carcinoma (RCC) tissues and cell lines [25]. The expression patterns of CASC2 showed close association with cancer progression, which might be a potential biomarker for management of cancers [26,27]. However, the clinical signi cance of CASC2 has been rarely investigated in bladder cancer.
In this study, we found that the expression level of CASC2 was signi cantly lower in bladder cancer patients than that in healthy volunteers. Moreover, the expression of CASC2 was negatively correlated with histological grade, TNM stage, and lymph node metastasis. The down-regulation of CASC2 might contribute to malignant progression of bladder cancer. The study performed by Li et al. indicated that the bladder cancer patients with low expression of CASC2 were more likely to undergo malignant disease progression and postoperative recurrence [28]. Pei er al. reported that the enforced expression of CASC2 could remarkably inhibit cell growth, migration and invasion, and promote cell apoptosis in bladder cancer cells in vitro. CASC2 might play anti-tumor action in bladder cancer through inhibiting Wnt/βcatenin pathway [14]. Both of the published articles might explain the results obtained in our study.
Given its distinct expression pro le in bladder cancer, we hypothesized that serum CASC2 might be a potential diagnostic biomarker for the cancer. Thus, ROC analysis was performed to detect the diagnostic value of CASC2 in bladder cancer patients. From the ROC curve, we could see that the CASC2 expression could discriminate between bladder cancer patients and healthy controls with the sensitivity of of 77.8%, speci city of 85.7%, and the AUC of 0.864. Serum CASC2 might be a potential non-invasive biomarker for early detection of bladder cancer. However, several limitations should be stated in current study. Firstly, the sample size was relatively small that might reduce the statistical power of our results. Second, the molecular mechanisms underlying the anti-tumor action of CASC2 in progression of bladder cancer was not explored in our study. In addition, all the individuals in control group were healthy, our results only con rmed that serum CASC2 could distinguish bladder cancer patients from the healthy individuals. Whether serum CASC2 could discriminate between bladder cancer patients and other malignancies cases remained unclear. Therefore, well-designed studies with a larger sample are required to verify and improve our conclusion.

Conclusions
In conclusion, serum lncRNA CASC2 is down-regulated in bladder cancer, and shows close association with aggressive cancer progression. Serum CASC2 may be a potential biomarker for early detection of bladder cancer.

List Of Abbreviations
Long non-coding RNAs (LncRNAs) 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.
Carcinoma of the Bladder. Medical science monitor: international medical journal of experimental and clinical research 2018, 24:438-447. Figure 1 CASC2 expression level between bladder cancer tissues and healthy tissues. The levels of CASC2 were signi cantly decreased in bladder cancer patients, compared to the healthy controls. Figure 2 ROC analysis was performed to estimate the diagnostic performance of serum CASC2 for bladder cancer patients.