Low Expression of hsa_circ_0018069 in Human Bladder Cancer and Its Clinical Significance

Abnormal expression of noncoding RNA molecules such as circRNA plays an important role in the development of malignant tumors. circRNAs are stable in structure and can be useful as ideal tumor markers. Advanced bladder cancer has poor treatment options and prognosis. Thus, we examined circRNAs to further understand the pathogenesis and development of bladder cancer and to identify molecular markers for the early diagnosis of bladder carcinoma. We found that hsa_circ_0018069 was differentially expressed in our RNA sequencing data. We used qRT-PCR to detect its expression in T24 and Biu-87 cell lines and in 41 paired samples of bladder cancer and adjacent normal tissue and analyzed the correlation between expression of hsa_circ_0018069 and the clinical characteristics of patients with bladder cancer. We then performed a bioinformatics analysis to reveal the mechanism of hsa_circ_0018069 in tumorigenesis of bladder cancer. The expression of hsa_circ_0018069 was significantly reduced in T24 and Biu-87 cells and was also significantly downregulated in bladder cancer tissues. Decreased expression of hsa_circ_0018069 was related to the grade stage (P=0.024), T stage (P=0.027), and muscular invasion depth (P=0.022) of bladder cancer. Bioinformatics analysis showed that hsa_circ_0018069 was coexpressed with protein-coding mRNAs that participate in cytoskeletal protein binding and cell-substrate junction assembly and play an anticancer role through focal adhesion and calcium signaling pathways. ceRNA analysis showed that hsa_circ_0018069 functions in ErbB, Ras, FoxO, and the focal adhesion signaling pathway by harboring miR-23c, miR-34a-5p, miR-181b-5p, miR-454-3p, and miR-3666. hsa_circ_0018069 may thus play an important role in the occurrence and progression of bladder cancer and serve as a valuable biomarker for the early diagnosis of this disease.


Introduction
Bladder cancer is one of the most common tumors in the urinary system [1]. Surgical operation is the main treatment for bladder cancer. However, when tumors advance to a high grade, the result of treatment is frequently unsatisfactory. Patients with high-risk bladder cancer may relapse, progress or die within 10 years [2]. The 5-year survival rate of high grade muscular invasive bladder cancer is only 6% [3]. Advanced bladder cancer displays poor sensitivity to radiotherapy and chemotherapy; therefore, early diagnosis of bladder cancer is particularly important.
In recent years, next-generation sequencing technology has been employed for transcriptome analysis and has provided valuable information on the diagnosis and underlying mechanisms of malignant carcinoma. With this technology, many noncoding RNAs have been discovered and studied, which has shown that abnormal expression of circRNA plays an important role in the formation of bladder tumors [4]. In our previous study [5], we found hundreds of circRNAs were up-or downregulated in bladder cancer tissues compared with adjacent tissues, of which hsa circ 0018069 was one of the most significantly downregulated circRNAs both confirmed by sequencing and real-time-PCR. It is spliced from chr10:30315031-30318795 with a length of 3764 nt and is formed by circularization of the fourth exon of KIAA . In the present study, we show that hsa circ 0018069 plays an important role in bladder cancer and may be useful as a biomarker for early diagnosis.

. . Expression Level and Clinical Relevance of hsa circ
. Based on the above data, we analyzed the correlation between the expression level of hsa circ 0018069 and clinical data of cancer patients. As shown in Table 2, downregulation of hsa circ 0018069 was significantly associated with grade stage (P=0.024), T stage (P=0.027) and muscular invasion depth (P=0.022), but was not associated with age, sex, tumor size or lymphatic metastasis.
. . Bioinformatics Analysis. We calculated Pearson's correlation coefficients among hsa circ 0018069 and differentially expressed mRNAs and selected genes with positive correlations >0.95 and negative correlations <-0.95 for further study. We performed GO (Figure 3(a)) and KEGG ( Figure 3(b)) analysis on coexpressed mRNAs with a group P value <0.05. Cytoskeletal protein binding and actin binding were the significant enriched GO terms, and focal adhesion and cGMP-PKG signaling were the main enriched pathways. We predicted miRNAs that could be sponged by hsa circ 0018069 and predicted targeted mRNAs based on the ceRNA mechanism. The mapped ceRNA network showed that hsa circ 0018069 could target AKT , SOS , PIK CB, PTEN, FOXO , DIXDC , and PPP R B by sponging miR-23c, miR-34a-5p, miR-181b-5p, miR-454-3p, and miR-3666 ( Figure 4). We also performed GO and KEGG analysis for the targeted mRNAs (Figures 5(a) and 5(b)) and showed that hsa circ 0018069 may play a tumor suppressor role through the ErbB, Ras, FoxO, and focal adhesion signaling pathways.

Discussion
Noncoding RNA was once considered meaningless for transcription, but recent studies have shown that it plays a vital role in the progression of disease [6]. CircRNA, without a 5' end cap and a 3' end poly-A tail, has a ring structure formed by covalent bonds and is resistant to exonuclease [7,8]. It is currently believed that circRNA is formed through a reverse splicing mechanism [9]. CircRNA can act as ceRNA, which is involved in the regulation of gene expression. It can also bind to proteins in the nucleus and modulate the function of transcription factors. Certain circRNAs have internal ribosome entry sites and may encode proteins. Low expression of hsa circ 002059 was confirmed to be related to overexpression of CEA and distant metastasis in gastric cancer [10], demonstrating that circRNA may contribute to clinical diagnosis and prognosis of tumors. However, the function of most circRNAs is not clear. In our sequencing data, we found that hsa circ 0018069 was significantly downregulated in four pairs of tumor and adjacent tissues, and we also examined the expression of hsa circ 0018069 in bladder cell lines and tissues by qRT-PCR. Results showed that in the T24 and Biu87 cell lines and in 33 out of 41 pairs of tissues, hsa circ 0018069 was downregulated, which indicates that hsa circ 0018069 plays an anticancer role in the occurrence and development bladder cancer.
Currently, most functions of circRNAs have been predicted by RNA-binding proteins or ceRNA mechanisms. In addition, we can also predict the function of circRNAs through correlated mRNAs. However, we found no RNAprotein binding sites in hsa circ 0018069 by online software. We therefore performed a coexpression and ceRNA analysis to elucidate the function of hsa circ 0018069. GO analysis of coexpressed mRNAs showed that the main enriched pathways were the focal adhesion and cGMP-PKG signaling pathways. The focal adhesion pathway was reported to be associated with embryonic development, tumor development and migration [11]. The genes enriched in the pathway include ACTN , FLNA, FYN, ILK, MYLK, PPP R B, PPP R C, and TLN . FLNA is associated with cell proliferation and invasion in hepatocellular carcinoma [12], while TLN participates in the invasion of ovarian serous carcinoma and is active in hepatocellular carcinoma cells [13,14]. We therefore deduced that hsa circ 0018069 may be involved in invasion of bladder cancer through the focal adhesion pathway. Another major enriched pathway was cGMP-PKG signaling, which plays an anticancer role in colon cancer [15] and renal cancer [16]. These two enriched pathways revealed that hsa circ 0018069 may act as an antioncogene in bladder cancer.
ceRNA analysis showed that hsa circ 0018069 could sponge miR-23c, miR-34a-5p, miR-181b-5p, miR-454-3p, and miR-3666. KEGG analysis of mRNAs showed enrichment in the ErbB, Ras, FoxO, and focal adhesion signaling pathways. In various kinds of malignant tumors, ErbB family members and partial ligands are frequently overexpressed, amplified, or mutated. For example, research revealed that EGFR was amplified and/or mutated in glioma and non-small-cell lung cancer [17], whereas ErbB was amplified in breast, ovarian and bladder cancer and in several other tumors [18,19]. ErbB receptor signaling regulates cell proliferation, migration, differentiation, apoptosis, and cell mobility in Akt, MAPK, and other pathways [20][21][22][23]. The Foxo signaling pathway is closely related to the insulin/PI3K/AKT signaling pathway and interacts with tumor suppressor gene P   [24,25]. FOXO signaling plays an inhibitory role in ovarian cancer, prostate cancer and colorectal cancer by inducing cell cycle arrest and apoptosis. In our study, hsa circ 0018069 was downregulated in bladder cancer tissues and exerts an anticancer effect, which suggested that hsa circ 0018069 may mediate the Foxo signaling pathway. Focal adhesion was a common pathway enriched in both KEGG pathway analysis of coexpressed mRNAs and in ceRNA analysis. The shared mRNA was PPP R B, which was significantly downregulated in bladder cancer in our sequencing result and in the TCGA database [26]. From our bioinformatics analysis, we speculate hsa circ 0018069 is involved in pathways related to tumorigenesis and development of bladder cancer mainly through sponging miR-181b-5p to suppress expression of PPP R B.
The diagnosis of bladder cancer is mainly confirmed based on the patient's symptoms, signs and clinical examination. Hematuria is a common symptom, but the incidence of gross hematuria in bladder cancer only accounts for 17% to 18.9% [27,28]. Ultrasound is valuable in diagnosis of bladder cancer, and transurethral bladder ultrasound shows high accuracy in tumor staging but is useless in the diagnosis of carcinoma in situ. Accuracy of diagnosis by CT and urine cytology is slightly lower, and interfering factors often lead to misdiagnosis. Currently, there is still no ideal biomarker that can replace cystoscope and urine cytology detection for bladder cancer and improve diagnosis, treatment, prognosis and postoperative follow-up [29]. Our research indicated that the accuracy of hsa circ 0018069 in the diagnosis of bladder cancer can reach 97.6%, with specificity of 46.3% and an AUC of 0.709. This indicated that hsa circ 0018069 may be valuable in the diagnosis of bladder cancer. The expression of hsa circ 0018069 was also correlated with T stage and muscular invasion, revealing that hsa circ 0018069 may serve as a preliminary marker to estimate tumor invasion. Combined with our bioinformatics analysis, we conclude that hsa circ 0018069 plays a vital role in the invasion of bladder cancer. The expression of hsa circ 0018069 can be combined with bladder microscopy results to determine clinical staging. Follow-up detection of hsa circ 0018069 can then be obtained from peripheral blood, which can reduce biopsy damage to patients and provide a new approach for the early diagnosis of bladder cancer.

Conclusion
In our study, hsa circ 0018069 was significantly downregulated and affected the T stage and invasion of bladder cancer. Our analysis suggested that hsa circ 0018069 played a significant role in the development of bladder cancer and may be useful as a biomarker for the early diagnosis of this disease.

Materials and Methods
. . Specimen and Clinical Data Collection. From October 2016 to October 2017, a total of 41 pairs of bladder cancer tissues and adjacent tissues were collected from the Department of Urology of the Fourth Affiliated Hospital of China Medical University (Shenyang, China). The adjacent tissues were more than 3 cm away from the tumor tissues. All patients included in the study were initially diagnosed without any treatments and were subjected to total or partial cystectomy. Diagnosis was confirmed by postoperative pathology. Bladder cancer was classified according to the World Health Organization international classification of oncology and TNM staging of the Union for International Cancer Control [30]. The experiment was approved by the Ethics Committee of China Medical University and all patients signed informed consent.
. . Sequencing Process and Analysis. Whole transcriptome sequencing was performed on four out of 41 pairs of bladder cancer tissues and adjacent tissues using the HiSeq X instrument. CIRCexplorer [31] was used to predict circRNAs. A criterion of |log2(fold-change)| ≥ 1, P value <0.05 and q <0.05 between two samples was used to identify differentially expressed genes and transcripts.
The primer sequences for the -actin internal reference were 5'-GAGACCTTCAACACCCCAGCC-3' (forward), 3'-GGATCTTCATGAGGTAGTCAG-5' (reverse). All primers were synthesized by Shanghai Shenggong Company (Shanghai, China). The PCR reaction conditions were set at 95 ∘ C for 5 s and 60 ∘ C for 34 s in a total of 40 cycles. The data was analyzed by ΔCT.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.