Urinary Exosomal Long Noncoding RNA TERC as a Noninvasive Diagnostic and Prognostic Biomarker for Bladder Urothelial Carcinoma

Purpose Bladder cancer is one of the most common urological malignancies worldwide, and approximately 90% of bladder cancer cases are histologically typed as bladder urothelial carcinoma (BLCA). Exosomes are 30 to 200 nm extracellular vesicles that transport microRNAs, long noncoding RNAs (lncRNAs), mRNAs, circular RNAs, and proteins across tissues and through circulation. Urinary exosomes may contain genetic information from tumor cells. Herein, we explored the clinical significance of urinary exosomal lncRNA telomerase RNA component (TERC) levels to provide an urgently needed diagnostic and prognostic biomarker for BLCA. Materials and Methods In this study, we used RNA-sequencing of samples from four BLCA patients and three healthy controls to identify that TERC was differentially expressed in urinary exosomes. We then used quantitative PCR in different types of clinical samples to validate the biomarker and analyzed results using receiver operating characteristic curves. Results We found that TERC was significantly upregulated in urinary exosomes from BLCA patients compared with those from healthy controls (P < 0.0001). Urinary exosomal TERC showed higher sensitivity (78.65%) and accuracy (77.78%) than existing indicators including nuclear matrix protein-22 and urine cytometry. Using the cut-off value 4.302, the area under the curve for urinary exosomal TERC was 0.836 (95% confidence interval: 0.768–0.891, P < 0.0001). Furthermore, this noninvasive assay could distinguish low-grade and high-grade tumors (P = 0.0153). Conclusions TERC is enriched in urinary exosomes from BLCA patients. Urinary exosomal TERC could become a diagnostic and prognostic biomarker for BLCA that allows clinicians to realize noninvasive detection of BLCA.


Introduction
Globally, bladder cancer is one of the most common urological malignancies, with approximately 90% of cases histologically defined as bladder urothelial carcinoma (BLCA). In 2020, the incidence of bladder cancer ranked tenth among all malignant tumors, and its incidence and mortality ranked second among male urinary system malignancies [1]. The gold standard for diagnosing bladder cancer remains tissue biopsy via cystoscopy. The colloidal gold immunochromatographic assay (GICA) for nuclear matrix protein-(NMP-) 22 and bladder tumor antigen are clinical methods approved by the US Food and Drug Administration; however, their sensitivity is too low, and hematuria always leads to false positive results [2][3][4]. Thus, there is an urgent need for a more meaningful diagnostic index for bladder cancer that balances sensitivity and specificity.
Exosomes are potential diagnostic samples from liquid biopsy because they can transport mRNAs, microRNAs, long noncoding RNAs (lncRNAs), circular RNAs, lipids, and proteins throughout bodily fluids [5,6]. The exosomal lipid bilayer protects the contents from RNases and proteases [7]. lncRNAs are defined as transcripts of >200 nucleotides and have been demonstrated to play critical roles in many biological and pathological processes rather than being "transcriptional noise" [8]. It has been reported that exosomes can selectively package certain lncRNAs that are abundantly present in exosomes [9,10]. BLCA tumor cells continuously release exosomes that contain lncRNAs into urine. Urinary exosomal lncRNAs can directly reflect the malignant state and have the possibility to be diagnostic and prognostic biomarkers for BLCA [11][12][13][14].
Using deep RNA-sequencing of urinary exosomes, we found that the lncRNA telomerase RNA component (TERC, 451 nt in length) was differently expressed between four BLCA patients and three healthy controls. TERC and telomerase reverse transcriptase (TERT) compose telomerase. In most human tumors, telomerase is activated to maintain telomere length, extend lifespan, and reduce apoptosis of tumor cells [15]. Many previous studies have reported the roles of TERC in telomere biology and in promoting tumorigenesis and inflammation, but there are still few reports regarding the diagnostic and/or prognostic value of exosomal TERC expression [16][17][18][19].

Isolation of Exosomes.
Exosomes for sequencing were isolated from 150 mL of urine using differential centrifugation. The isolation procedure was as follows, taking the supernatant from each step: urine was centrifuged at 600 × g for 20 min, 2000 × g for 30min, and 10000 × g for 1h and then filtered through a 0.22 μm filter. Finally, ultracentrifugation at 100000 × g for 2h was performed, and the supernatant was discarded. All centrifugation steps were performed at 4°C. Exosome pellets were resuspended in 50 μL of 0.22 μm filtered phosphate-buffered saline.
Exosomes used for validation were isolated from human urine in strict accordance with the instructions of the exosome extraction kit (BestBio, Shanghai, China). 8 mL of urine was centrifuged at 3000 × g for 15 min at 4°C, and the supernatant was then centrifuged at 10000 × g for 20 min at 4°C. The supernatant was then mixed with solution A and stored 4°C overnight. The mixture was centrifuged at 10000 × g for 1 h at 4°C and exosome pellets were resuspended in 500 μL TRIzol-LS (Life Technologies, Carlsbad, CA, USA).

Transmission Electron Microscopy (TEM).
Exosome suspensions were placed on 400-mesh carbon-coated copper grids and negatively stained with 2% phosphotungstic acid solution. TEM was performed to view and capture images (Thermo Fisher Scientific, Waltham, MA, USA).
2.4. Nanoparticle Tracking Analysis (NTA). The NanoSight LM10 system (Malvern Instruments Ltd., Malvern, UK) was used to detect the concentration and size distribution of particles.

Western Blot Analysis.
Exosome suspensions were mixed with the appropriate volume of 5x SDS loading buffer, the mixture was boiled for 15 min at 100°C, and then, the supernatant was collected for western blot analysis. The primary antibodies included anti-TSG101 (Abcam, Cambridge, UK), anti-HSP70, anti-Annexin V, and anti-CD9 (all from Cell Signaling Technology, Danvers, MA, USA). The secondary antibody used was peroxidase-conjugated goat antirabbit IgG (Millipore, Burlington, MA, USA).
2.6. RNA-Sequencing. RNA-sequencing (RNA-seq) was performed by Shanghai Kangcheng Biotechnology Company (Shanghai, China) using an Illumina Novaseq 6000 system (San Diego, CA, USA). Differences in transcript levels were analyzed and used to screen out the differentially expressed lncRNAs between the BLCA patient and healthy control groups. The comparison scheme was fold change > 1:5, P value (F test) ≤ 0.05, and mean FPKM ≥ 0:5 in each sample.
2.7. RNA Extraction and Real-Time Quantitative PCR (qPCR). TRIzol-LS reagent was used to extract total exosomal RNA, cDNA was synthesized using the Prime-Script™ RT reagent kit with gDNA Eraser (Takara, Dalian, Japan), and qPCR was performed using the TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara) on an Applied Biosystems 7300 real-time PCR system (Waltham, MA, USA). Table 1 shows the sequences of primers used in this study.
2.8. GICA and Enzyme-Linked Immunosorbent Assay (ELISA) Analyses of NMP-22. Urine exosomal NMP-22 was detected using the Alere NMP22® BladderChek® Test based on GICA and ELISA kits from Hualian Biotechnology (Wuhan, China). The assays were performed in accordance with the kit instructions.
2.9. Statistical Analysis. Data are presented as mean ± SD. The Mann-Whitney U test, Pearson's χ 2 test, and Pearson's correlation analysis were used to assess differences or

Identification of Exosomes Derived from Urine Samples.
According to the 2018 checklist for minimum information for studies of extracellular vesicles (MISEV2018) [20], we used a combination of TEM, NTA, and western blot analysis to identify exosomes. TEM showed that urinary exosomes were of round shape with cup-like concavity (Figure 1(a)). The size distributions of exosomes were analyzed by NTA. As shown in Figure 1(b), the size (diameter) of urinary exosomes ranged from approximately 50 to 200 nm (mean: 165.2 nm). Positive expression of four exosomal protein markers was confirmed by western blot analysis ( Figure 1(c)) including of two cytosolic proteins (TSG101 and Annexin V) and two transmembrane proteins (HSP70 and CD9). Only strictly identified urine exosomes were used for further analysis and research [21].

RNA-Sequencing of Urinary Exosomes.
To identify differentially expressed BLCA-specific molecules, we performed RNA-sequencing on urine exosomes from four BLCA patients and three healthy controls [22]. The results revealed 106 differentially expressed lncRNAs between urine exosomes from BLCA patients and healthy donors, among which 103 were upregulated and three were downregulated (Figures 2(a) and 2(b)). Among the upregulated lncRNAs, we found high abundance of TERC, which has been previously reported to be related to "tumor growth."

Increased TERC Expression in Urine
Exosomes from BLCA Patients. We performed qPCR to evaluate relative TERC expression in different types of clinical samples. We found that 18S levels were more stable and abundant in urinary exosomes than the conventional housekeeping gene GAPDH and was more suitable as an internal reference gene for lncRNAs in exosomes. The absolute expression levels of genes are listed in Supplementary File 2. First, we detected TERC expression in urine exosomes from the three healthy controls and four BLCA patients used for sequencing ( Figure 3(a)). In this small sample, we verified that TERC expression was higher in urinary exosomes from BLCA patients than from healthy controls, which was consistent with the RNA-sequencing results. TERC was then detected in urine exosomes from 94 healthy controls, 46 patients with urinary benign lesions, and 128 BLCA patients (Figure 3(c)). The findings suggested that urinary exosomal TERC expression in BLCA patients was significantly higher compared with that in healthy controls and patients with urinary benign lesions (P < 0:0001), indicating that urinary exosomal TERC may have diagnostic value for BLCA. As shown in Figure 3(b), there was no difference between TERC expression of tumors (n = 39) and that of adjacent tissues (n = 23).

Urinary Exosomal TERC Had Better Diagnostic
Value than NMP-22 (ELISA and GICA) and Urine Cytology. We examined TERC levels in urine exosomes from 89 BLCA patients and 63 healthy controls to determine its diagnostic utility. ROC curve analysis using the cut-off value 4.302 showed that TERC was a promising diagnostic biomarker with an area under the curve (AUC) of 0.836 (95% confidence interval [CI]: 0.768-0.891, P < 0:0001). The sensitivity and specificity of TERC were 78.65% and 77.78%, respectively (Figure 4(a)). Meanwhile, we detected NMP-22 from urine samples using ELISA; the results are listed in Supplementary File 2. The AUC, sensitivity, and specificity of NMP-22 (ELISA) were 0.696 (95% CI: 0.616-0.768, P <         (Figures 5(a) and 5(b)). These results indicated that urinary exosomal TERC was a significant risk predictor with the potential to be a prognostic biomarker for BLCA. a P < 0:05, Pearson's χ 2 test for the variables above in patients.

Urine Exosomal TERC Expression Was Not Correlated with Paired Tissues, and It Stably Exists after Freeze-Thaw
Cycles due to Protection from the Lipid Bilayer. To investigate the relationship in TERC expression between different types of samples, TERC levels were measured by qPCR in paired tissues and urine exosomes from BLCA patients [23]. Pearson's correlation analysis demonstrated that urine exosomal TERC levels were not correlated with the TERC levels in corresponding tissues; the correlation coefficients (r) were 0.00954 (Figure 6(a)).
Additionally, we divided urine samples into five aliquots of equal volume and placed each at −80°C for one to five freeze-thaw cycles [24]. The results revealed that exosomal TERC levels were stable after multiple freeze-thaw cycles (Figure 6(b)). This suggested that storing urine at −80°C even with multiple freeze-thaw cycles will not affect exosomal TERC, which was superior to detecting TERC in urine sediment cells [25,26]. In summary, urinary exosomal TERC has the potential to be a stable and noninvasive diagnostic and prognostic biomarker for BLCA.

Discussion
Bladder cancer is one of the most frequent malignancies of the urinary system, with 90% of cases pathologically classified as BLCA. The cause of BLCA tumorigenesis is currently unclear and may include smoking, repeated infection, exposure to compounds, or genetic factors [27,28]. The gold standard for diagnosing bladder cancer is pathological tissue biopsy, which is invasive and uncomfortable. This study introduced a novel assay that does not require expedited or other special handling procedures, and an 8 mL urine sample is more than enough for the analysis.
We identified that the lncRNA TERC was differentially upregulated in urinary exosomes of BLCA patients by RNA-sequencing. The important components of the telomerase are TERT and TERC. As previously reported, identifying TERT promoter mutations in the urine may substantially aid in the early detection of bladder cancer [29,30]. However, exosomal TERC has thus far not been investigated as a diagnostic or prognostic biomarker for bladder cancer.
We found that TERC expression levels were not significantly different between tumor and adjacent nontumor tissues from BLCA patients but were significantly upregulated in urinary exosomes from BLCA patients compared with those from healthy controls. Furthermore, urinary exosomal TERC expression could distinguish between urinary benign diseases (inflammation, stones, and obstruction) and bladder cancer (P < 0:0001). TERC can circulate in whole body fluids under the envelopment of exosomes. Additionally, TERC could significantly distinguish high-versus low-grade BLCA (P = 0:0153), and Kaplan-Meier survival curves demonstrated its prognostic value. Importantly, multiple freeze-thaw cycles of urine did not affect the stability of exosomal TERC.
The exosome is the star molecule of liquid biopsy, and research into them is still in the early stages. Our findings are limited by current science and technology in terms of achieving rapid and efficient detection of exosomal RNA. To extract urinary exosomes, we employ the polyethylene glycol (PEG) precipitation technique, which normally involves around 10 hours of pretreatment (centrifugation and resting) and 1 hour of centrifugation at 10000 × g, and the exosome precipitation will be achieved using a standard high-speed centrifuge. Although costly ultracentrifuges are not needed, they still have the drawback of being timeconsuming and multistep. At this stage, urine exosomal TERC can only be used as an adjunct to cystoscopy, with diagnostic efficacy superior to existing NMP-22, BTA, and other indicators, allowing for patient review and follow-up. From experimental research to clinical application, not only medical but also engineering, science, and other interdisciplinary collaborations are required, and completely automated batch detection of urine exosomal TERC is still a long way off.
In the future, more data on the detection of urine exosomal TERC will be collected via multicenter and large sample studies, and patients will be followed for a longer period of time to validate its diagnostic and prognostic value.

Conclusions
In summary, the long noncoding RNA TERC was first found in urine exosomes in this article, and we demonstrated that urinary exosomal TERC can be used as a diagnostic and prognostic biomarker for BLCA with the added benefit of reducing unnecessary injuries from tissue biopsies. This study provides a convenient, stable, repeatable, and noninvasive index for clinicians and BLCA patients. Thus, our discovery will have profound implications for diagnosis and monitoring of bladder urothelial carcinoma.

Data Availability
The datasets during and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.