Circular RNA CCT3 is a unique molecular marker in bladder cancer

This study surveyed circular RNA CCT3 in bladder cancer (BCa). We recruited 85 BCa patients and 40 normal controls (Normal) and collected clinical specimens for analysis. circRNA CCT3 expression was analyzed by RT-qPCR, diagnostic accuracy was calculated by ROC curves, and survival outcomes were evaluated by survival curves. CircRNA CCT3 was overexpressed or knocked down in cells, thereafter to observe the changes in cell malignant phenotypes. The downstream molecules of circRNA CCT3 were detected. Our data suggest that circRNA CCT3 was upregulated in human BCa and was associated with poor survival outcomes of BCa patients. In cell experiments, overexpressing circRNA CCT3 promoted BCa cell malignancy, whereas silencing circRNA CCT3 did the opposite. In addition, circRNA CCT3 modulated PP2A expression by miR-135a-5p. This study demonstrates that circRNA CCT3 is a diagnostic and prognostic biomarker in BCa patients and is a tumor promoter in BCa. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-023-11510-0.


Introduction
The most common subtype of bladder cancer (BCa) is urothelial carcinoma or transitional cell carcinoma, accounting for more than 90% of all cases [1].In 2020, BCa ranked 9th in incidence globally and 13th in cancerrelated deaths among all cancers [2].BCa can be divided into two groups according to their different behaviors: high-grade muscle-invasive BCa (MIBC) and low-grade non-muscle-invasive BCa.Surgery is the main treatment for BCa.However, the advanced disease often progresses to MIBC for which the treatment is often suboptimal [3,4].This is because MIBC often metastasizes to lymph BCa and influence the progression of BC has not yet been demonstrated.
It is well known that the main function of circRNA is to function by binding to miRNA [18] Based on bioinformatic analyses, miR-135a-5p was identified as binding to circRNA CCT3.miR-135a-5p acts as a cancer suppressor in a variety of cancers, such as non-small cell lung cancer [19] and breast cancer [20].We found the binding sequence of miR-135a-5p in the protein phosphatase 2 A (PP2A) 3'UTR through bioinformatic analyses.PP2A has been implicated in many cancer types including BCa [21][22][23].We speculated that circRNA CCT3 mediates BCa cell progression and acts as a potential regulator of PP2A and aimed to provide new perspectives on BCa pathogenesis and a new theoretical basis for the targeted therapy of BCa.

Clinical sample
From December 2014 to May 2018, urine samples from 85 patients with BCa and 40 healthy subjects were obtained from The First Affiliated Hospital of Shaoyang University.These urine samples were taken from the subjects' morning urine.Urine was collected in a sterile environment and that only midstream urine was stored for analysis.For each patient, 25 mL of urine was collected for analysis.The urine was quickly stored at -80 °C to avoid RNA degradation.All clinical experiments were conducted following the principles of The First Affiliated Hospital of Shaoyang University Ethics Committee.Written consent has been obtained from all subjects.

Patient's information
Clinicopathological data included age, gender, multiple, tumor size, tumor stage (Union for International Cancer Control stages), lymph node metastasis, and pathological grade (2004 World Health Organization urothelial tumor classification) were obtained.All patients were followedup until April 30, 2020, during which the recurrence-free survival (RFS) and overall survival (OS) were calculated [15].

Colony formation experiment
Cells (6000 cells/well) were maintained in 10% FBS-DMEM for 8 d and the number of colonies was counted after 0.1% crystal violet staining [10].

Migration and invasion analysis
Cells (1 × 10 5 ) were suspended in 200 µl of serum-free medium and transferred into the upper chamber of transwell (8 μm, Costar), whose bottom chamber contained 10% FBS medium.After 2 h, lower surface cells after 0.1% crystal violet staining were seen under a microscope (Olympus, Japan).Matrigel (BD Biosciences) was only utilized in invasion analysis [8].

RNA extraction and analysis
Total RNA was extracted by Trizol (15596-018, Invitrogen) and reverse transcribed into cDNA by Primescript™ RT reagent (RRO37A, TaKaRa) or Mir-X miRNA First Strand Synthesis Kit (638,315, Clontech, USA).PCR was implemented with SYBR Premix Ex TaqTM II Kit (TaKaRa) in an ABI PRISM 7300 system.U6 and GAPDH served as internal controls.The primers shown in Table 1 were synthesized by Bio Just (Wuhan, China). 2 −ΔΔCt was the gene calculation formula [25].

Statistical analysis
All data were presented as mean ± standard deviation and evaluated by SPSS 20.0.Two sets of data were assessed by paired Student's t-test or unpaired Student's t-test while multiple sets of data were by ANOVA and Dunnett's test.Chi-square test compared the categorical data, and ROC curve evaluated the diagnostic accuracy.Kaplan-Meier curve and log-rank test assessed the OS and RFS in patients with BCa, and Cox regression proportional hazard analysis examined prognostic factors.P < 0.05 represented significant differences.

circRNA CCT3 is highly expressed in BCa
To discover potential BCa-related circRNAs, urine samples from 8 healthy controls and 8 BCa patients were analyzed by microarray, and 21 circRNAs were upregulated and 14 were downregulated in BCa patients (Fig. 1A).Among the most significantly changed circRNAs, hsa_circ_0004680 was further filtered (Fig. 1B).hsa_ circ_0004680 is located at chr1:156303337-156,304,709, and the host gene is CCT3, with 1372 bp genome length and 211 bp spliced length (Fig. 1C).To further evaluate the potential significance of circRNA CCT3, its expression was checked in 40 cases of healthy controls and 85 cases of BCa samples.It was discovered that circRNA CCT3 expression was elevated in BCa patients (Fig. 1D), suggesting that circRNA CCT3 is highly expressed in BCa and may be associated with tumor development.

Diagnostic value of circRNA CCT3 in BCa patients
To evaluate the diagnostic value of circRNA CCT3 in BCa patients, ROC analysis was employed to analyze the role of plasma circRNA CCT3 in distinguishing BCa patients from normal controls.The data presented that  the optimal diagnostic cutoff value of circRNA CCT3 was 1.09, and the AUC value was 0.8575 (95% CI, 0.734-0.896)(Fig. 2), the sensitivity was 79.5% and specificity was 65.6% (Table 3).This suggests that circRNA CCT3 in plasma has potential diagnostic value for BCa.

Inhibition of circRNA CCT3 depresses the growth of BCa cells
Next, circRNA CCT3 in BCa tumor progression was further investigated in cells.Consistent with online database analysis, circRNA CCT3 expression was upregulated in T24 cell line compared to SV-HUC-1 cell line (Fig. 4A).Subsequently, siRNA and overexpressed plasmids targeting circRNA CCT3 were transfected into T24 cells.qPCR results confirmed successful knockdown and overexpression of circRNA CCT3 (Fig. 4B).CCK-8 and colony formation experiments revealed that overexpressed circRNA CCT3 further increased the proliferation rate and number of cloned cells in T24 cells, while knocking down circRNA CCT3 repressed T24 cell proliferation (Fig. 4C, D).Transwell assay results indicated that overexpressed circRNA CCT3 forced cell migratory and invasive capacities, while downregulated circRNA CCT3 did the opposite (Fig. 4E, F).Also, flow cytometry showed the effect of circRNA CCT3 on apoptosis of T24 cells.Elevating circRNA CCT3 increased the apoptosis rate of T24 cells, while knocking down circRNA CCT3 enhanced cellular apoptosis (Fig. 4G).In conclusion, inhibiting circRNA CCT3 reduced the malignant progression of BCa cells.

circRNA CCT3 promotes malignant behavior of BCa cells by regulating the miR-135a-5p/PP2A axis
The role of miR-135a-5p/PP2A axis in the regulation of BCa by circRNA CCT3 was analyzed through functional rescue experiments.Overexpression of circRNA CCT3 was accompanied by overexpression of miR-135a-5p or knockdown of PP2A, respectively.RT-qPCR experiments showed that overexpression of circRNA CCT3 inhibited miR-135a-5p and promoted PP2A expression, but this effect was reversed by overexpression of miR-135a-5p or knockdown of PP2A, respectively (Fig. 6A, B).CCK-8 and colony formation experiments showed that the promotion effect of circRNA CCT3 on the proliferation of T24 cells was reversed by overexpression of miR-135a-5p or PP2A knockdown (Fig. 6C, D).Transwell experiments showed that overexpression of circRNA CCT3 could promote the invasion and migration ability of T24 cells, but overexpression of miR-135a-5p or PP2A knockdown could reverse this phenomenon (Fig. 6E, F).In addition, the inhibitory effect of overexpression of circRNA CCT3 on the apoptosis rate of T24 cells was reversed by overexpression of miR-135a-5p or PP2A knockdown (Fig. 6G).These data suggest that circRNA CCT3 affects the malignant behavior of BCa cells by regulating the miR-135a-5p/PP2A axis.

Discussion
BCa is one of the most common tumors of the urinary system, with high morbidity and mortality worldwide [28].The prognosis of patients with advanced BCa is poor, so early diagnosis and the search for reliable and effective therapeutic targets are needed [29].Many cir-cRNAs have been shown to be abnormally expressed in BCa, and they are not only involved in regulating various biological behaviors of BCa, but also some circRNAs can be biomarkers for the diagnosis of BCa [30,31].However, the current understanding of circRNA as a biomarker and cancer factor for the early diagnosis of BCa is not comprehensive.This study analyzed clinical samples from BCa patients and screened out significant differentially expressed circRNA molecules.Among these abnormally expressed circRNAs, circRNA CCT3 (hsa_circ_0004680) was particularly studied.It was highly expressed in BCa and mediated BCa cell activities via miR-135a-5p/CCT3 axis.In addition, circRNA CCT3 in plasma was found to be a potential biomarker for BCa diagnosis.Diagnostic biomarkers are of great significance for the early screening and malignant differentiation of BCa [32].Since blood samples are most readily available to patients during early screening, blood biomarkers are important for the diagnosis of BCa [33].Although many circRNAs have been confirmed to be involved in various biological behaviors of BCa cells, few can be considered diagnostic biomarkers.This may be due to the large abnormalities in the abundance of different circRNAs in cancer tissues and blood.This study found that circRNA CCT3 was highly expressed and had a high abundance in both BCa tissues and blood of BCa patients.High levels of circRNA CCT3 were confirmed by ROC curve as a potential diagnostic biomarker for BCa.Although previous studies have confirmed abnormal expression of circRNA CCT3 and its involvement in cancer development [34,35], this study is the first to demonstrate the potential value of circRNA CCT3 in cancer diagnosis.Furthermore, circRNA CCT3 was associated with poor outcomes in BCa patients.The reason can be found in cell experiments, demonstrating that overexpressed circRNA CCT3 can promote proliferative, invasive, and migratory capacities of BCa cells and inhibit apoptotic capacity.It is speculated that high levels of circRNA CCT3 will accelerate the deterioration of BCa and thus reduce the survival time of patients with BCa.Notably, two previous studies have also shown that circRNA CCT3 promotes distal metastasis of cancer cells and EMT [16,34].It is speculated that circRNA CCT3 may also have similar biological functions in BCa, but it needs to be verified in subsequent xenotransplantation modelsSubsequently, the dual luciferase reporter assay, RNA-pull down assay, and RIP assay confirmed that cir-cRNA CCT3 mediated PP2A by competitive adsorption of miR-135a-5p.It is speculated that the miR-135a-5p/ PP2A axis may be one of the downstream pathways that circRNA CCT3 plays the role of oncogene in BCa.miR-135a-5p acts as a cancer suppressant in various cancers [20,36,37].This study confirmed that miR-135a-5p is lowly expressed in BCa, but it is not clear whether it is a cancer suppressor in BCa, which needs to be verified in future studies.In addition, it is necessary to explore the role of circRNA CCT3/miR-135a-5p/PP2A in the biological behavior of BCa through functional rescue studies.This will provide data support for the search of potential therapeutic targets for BCa.

Conclusion
In conclusion, this study demonstrated the specific high expression of circRNA CCT3 in BCa.circRNA CCT3 expression is closely related to the malignancy of BCa and the prognosis of BCa patients.These results suggest that circRNA CCT3 has a potential value in the diagnosis and treatment of BCa as a unique molecular marker.In addition, signaling pathways associated with circRNA CCT3 in BCa are discovered, which provide new ideas and directions for further research and treatment of BCa.

Fig. 2
Fig. 2 Diagnostic value of circRNA CCT3 in BCa patients.ROC curve showed the discriminative ability of plasma circRNA CCT3 in BCa patients and normal controls

Table 1
Primer sequences in PCR

Table 2
Baseline characteristics of bladder cancer patients Note: SD, standard deviation

Table 3 ROC
curve analysis of the diagnostic value of plasma circRNA CCT3 in bladder cancer

Table 5
Univariate and multivariate analysis of prognostic factors associated with recurrence-free survival