Circular STAG2 RNA Modulates Bladder Cancer Progression via miR-145-5p/TAGLN2 and Is Considered as a Biomarker for Recurrence

Simple Summary Bladder cancer (BCa) is a major urologic malignancy with a high potential for death-threatening complications. Successful treatment relies on timely diagnostics and prediction of clinical outcomes. Molecular markers can be helpful for earlier diagnosis and for making treatment decisions. The biomarker landscape for BCa is not satisfactory yet. The discovery of novel diagnostic indicators for making treatment decisions is critical. Biological fluids are the most convenient and reliable source of these molecules. Circular RNAs (CircRNAs), special non-coding RNAs with a stable structure that are formed by a unique backsplicing process, recently became popular candidates for biomarkers in cancer biology. CircRNAs are widely distributed within the body including biological fluids. Their biological functions include gene regulation via sponging microRNAs, another type of small regulatory RNA. Our study characterized the circRNA hsa_circ_0139697 (circSTAG(16–25)) derived from exons 16 to 25 of STAG2, a gene known to play an important role in BCa biology that can serve as a biomarker predicting recurrence. We found that circSTAG2(16–25) could promote the proliferation, migration, and invasion of BCa cells by binding miR-145-5p, which causes the upregulation of the potential onco-gene TAGLN2. In addition, circSTAG(16–25) can serve as a biomarker for recurrence prediction. Abstract The current study aimed to elucidate the regulatory mechanisms of the circRNA hsa_circ_0139697 (circSTAG2(16–25)) in BCa and to consider the opportunity of using circSTAG2(16–25) isolated from BCa patient urine as a marker for disease development prediction. The selection of this circRNA was determined by the special role of its parental gene STAG2 in BCa biology. The circRNA hsa_circ_0139697 was chosen from 25 STAG2 circRNAs due to its differential expression in the urine of BCa patients and healthy volunteers. Higher levels of circSTAG2(16–25) were detected in urine samples obtained from patients with recurrent tumors. A higher expression of circSTAG2(16–25) was also detected in more tumorigenic BCa cell lines. The overexpression of circSTAG2(16–25) in BCa cells induced the elevation of proliferation, motility, and invasion. To study the mechanisms of circSTAG2(16–25) activity, we confirmed that circSTAG2(16–25) can bind miR-145-5p in vitro as was predicted by bioinformatic search. miR-145-5p was shown to suppress some genes that promoted BCa progression. One of these genes, TAGLN2, encodes the protein Transgelin 2, which plays a role in BCa cell motility and invasion. Therefore, the possible mechanism of action of circSTAG2(16–25) could be sponging the tumor suppressor miR-145-5p, which results in activation of TAGLN2. In addition, circSTAG2(16–25) might be considered as a potential biomarker for recurrence prediction.


Introduction
Bladder cancer is the second most common malignancy of the urinary system, and it is a heterogeneous disease with significant diagnostic, therapeutic, and prognostic problems.Early detection signifies a better disease prognosis; thus, minimally invasive diagnostic options are needed to improve patient outcomes [1].Molecular markers can provide the opportunity to diagnose a bladder tumor early, identify patients who are at risk of recurrence, and/or predict how tumors will respond to therapeutic approaches [2].A patient's urine can be considered a reliable and convenient source of such biomarkers.DNA, RNA and protein molecules derived from the urine of BCa patients have previously been considered as diagnostic and prognostic markers for BCa [3].
Circular RNAs (circRNAs), which have covalently linked ends, have attracted increasing attention because of their potential for biological activity in different clinical fields, including cancer biology [4,5].It was shown that circRNAs isolated from urine can serve as biomarkers for different urological diseases [6].CircRNAs are generated during pre-mRNA splicing when a downstream 5 ′ splice site is joined to an upstream 3 ′ splice site [7][8][9].Most mature circRNAs are expressed at low levels, but some have known physiological functions including in gene regulation, e.g., by binding to microRNAs, and/or accumulating to higher levels than their associated linear mRNAs [10].This is because circRNAs are naturally resistant to riboexonucleases and more stable than linear RNA molecules, making them potentially very promising biomarkers.One possible mechanism of circRNA action is sponging or sequestering another type of active small RNA, microRNA.MicroRNAs suppress the activity of mRNAs that regulate the progression of multiple cancers [4,11].
For this study, we focused on a circRNA synthesized from the human STAG2 gene that played a perceptible role in BCa biology.Stromal antigen 2 (STAG2) protein encoded by the STAG2 gene is a subunit of the cohesin complex, which regulates sister chromatid separation during cell division.STAG2 is one of the most mutated genes in BCa.Most mutations (non-sense, frameshift, and splicing) are inactivating, and induce complete loss of protein expression in non-muscle invasive BCa (NMIBC).Published data suggest that the loss of STAG2 production is a good prognostic factor in NMIBC [12][13][14][15][16][17][18].

Patient Specimens
Eighty urine samples and seven matched tumors were collected from patients diagnosed with bladder cancer from March 2022 to September 2023.Twenty additional urine specimens were obtained from healthy donors from March 2022 to August 2023.Fifty-three examined patients were male.The age range was 53-95 years.The pathological types were confirmed by professional pathologists.Written informed consent was acquired from all patients, and this study was approved by the IRB committee of Stony Brook School of Medicine.

RNase R Treatment
Total RNA was incubated for 30 min at 37 • C with or without 3 U/µg RNase R (Epicentre Technologies, Mira-Bhayandar, India) followed by RNase R inactivation at 65 • C for 20 min.K + was replaced with Li + in the reaction buffer to improve the efficiency of linear RNA degradation [26].RNA was then purified using miRNeasy Mini Kit (QIAGEN, Germantown, MD, USA) and eluted in 15 µL of water.

Wound-Healing Assay
Wound-healing assays were performed as previously described [28].Transfected and control cells were cultured to 85-95% confluency in 24-well plates.The scratch was made using a 200 µL tip.Using a microscope camera, photos of the cell scratches were taken at 0 and 24 h.Measurements of the wounded area were performed with Image J software (version 1.54h).

Transwell Invasion Assay
For the Transwell invasion assay, 200 µL serum-free culture medium containing 2 × 10 4 cells was plated into the upper chamber of a 24-well plate, which was precoated with Matrigel (BD Biosciences, San Jose, CA, USA).600 µL medium containing 10% FBS was then added to the lower chamber.After incubation for 24 h, the cells that migrated to the membrane of the upper chamber were fixed with 4% paraformaldehyde and stained with 1% crystal violet.The invasive cells were treated with 10% acetic acid and spectrometric absorbance at 525 nm was determined with plate reader [29].

Statistical Analysis
Experimental data are shown as mean ± standard deviation (SD).Student's t-test was performed to compare differences between unpaired groups.Experimental data were analyzed by GraphPad Prism 5.0.1 software.p-value < 0.05 was considered statistically significant.

CircSTAG2(16-25) Is Up-Regulated in BCa and in More Aggressive Urothelial Carcinoma Cell Lines
The STAG2 gene is highly mutated in BCa (23%).In most NMIBC cases, mutations cause STAG2 to be truncated and these genomic alterations are associated with low recurrence rates [13,16,18,31].There are no publicly available expression data on circular RNAs derived from the STAG2 gene in BCa or the human urothelium.We thus selected STAG2 for circRNA synthesis and evaluation because of its highly mutated status and specific role in BCa.Twenty-six distinct circRNAs derived from the STAG2 gene have been annotated in circBase [32].To determine if some of these circRNAs could be detected in urine, we used RT-qPCR and divergent primers that amplify across the back splicing junction to test the expression of 9 of these circRNA candidates.These candidates were selected based on their mature transcript size and their potential ability to bind miRNAs that showed activity in BCa.All tested circSTAG2 transcripts were expressed at some level in the urine of both BCa patients and healthy volunteers, with some showing similar expression levels in patients and controls while others appeared differentially expressed, but the difference was not always statistically confirmed (Figure 1A).One circRNA-circSTAG2 (16)(17)(18)(19)(20)(21)(22)(23)(24)(25), also known as hsa_circ_0139697, a 1117 nt transcript that has the end of exon 25 back spliced to the beginning of exon 16 was selected for further study (Figure 1B).This circRNA was selected because its expression was significantly increased in urine derived from BCa patients compared to the urine obtained from control subjects.
We further measured circSTAG2(16-25) levels using RT-qPCR in several urothelial cell lines and found that its expression levels were higher in 5637 and T24 cells, which are aggressive BCa cell lines, compared to non-transformed urothelial cells (UROtsa) (Figure 2C).Another circRNA derived from STAG2 (hsa_circ_0091460) was used as a control to compare expressions.As expected, circSTAG2 (16)(17)(18)(19)(20)(21)(22)(23)(24)(25) was resistant to digestion by the exonuclease RNase R, unlike linear RNAs like U6 snRNA and STAG2 mRNA that were, at least partly, digested (Figure 2D).To examine circSTAG2 (16)(17)(18)(19)(20)(21)(22)(23)(24)(25) expression relative to clinical status, we compared its expression in urine from patients with different clinical histories.The clinical course of each bladder cancer patient was reviewed.Patient clinical courses were separated into remission, recurrence, or progression categories.Remission was defined as the absence of bladder cancer while patients remained on surveillance status post-surgery for bladder cancer.Recurrence was defined as the identification of bladder cancer of equal or lower grade and stage on surveillance after initial bladder cancer treatment.Progression was defined as the upstaging of bladder cancer to a higher grade or stage on subsequent surveillance.Bladder cancer surgeries were performed in the standard transurethral technique.Ten samples (age 55-70) were selected from each group.CircSTAG2(16-25) was detected at ~5× fold higher levels in urine derived from patients with recurrent tumors as compared with healthy samples.It was ~2.0× fold higher in urine derived from patients with remission and ~1.7× fold higher in urine samples derived from patients with potential BCa progression (Figure 2E).

miR-145-5p Expression Is Correlated with BCa Progression and It Suppresses Proliferation, Migration, and Invasion of BCa Cells
Using RT-qPCR, we found suppressed expression of miR-145-5p in the urine of BCa patients and in more aggressive BCa cell lines (Figure 5A,B).The 5637 and T24 cell proliferation activity was modulated by miR-145-5p expression (Figure 5C,D).Mobility of 5637 (Figure 5E,F) and T24 cells (Figure 5I,J) was dependent on the levels of miR-145-5p.For
Animal study is an important part of cancer biology research.There are several types of BCa animal models that can be developed in rodents.These models include (i) orthotopic (the tumor grows in the urothelium) and (ii) heterotopic (the tumor grows outside the bladder).In addition, based on the type of tumor-forming cells, models can be divided into three types: (a) xenogeneic (implantation of human bladder cancer cells or tissue into immunodeficient mice), (b) syngeneic (implantation of murine cells), and (c) transgenic (genetic modification of experimental animal) [40].In this study, we were interested in a human circular RNA isolated from urine of BCa patients that originated from BCa tumors, so the orthotopic xenogeneic model is the only logical option to support the concept of this study.Heterotopic models do not provide direct contact of the tumor with urine, and the circular RNA of interest may not be produced in an orthotopic mouse model.It is possible to establish orthotopic implantation of human BC cultured cells in the bladder wall [41].The tumor grows relatively fast, and cells may be modified before implantation.Future results related to human circSTAG2 (16)(17)(18)(19)(20)(21)(22)(23)(24)(25) biology might thus be obtained by using cultured BC cells in this model, but it is generally much more complicated to implant and grow human BC tissue in rodent urothelium.
using cultured BC cells in this model, but it is generally much more complicated to implant and grow human BC tissue in rodent urothelium.

Figure 1 .Figure 1 .
Figure 1.Identification and characterization of circSTAG(16-25) (hsa_circ_0139697) selected for this study.(A) Expression of nine selected circRNAs in the urine of three healthy volunteers and three Figure 1.Identification and characterization of circSTAG(16-25) (hsa_circ_0139697) selected for this study.(A) Expression of nine selected circRNAs in the urine of three healthy volunteers and three BCa patients (age-matched, two males, one female).* = p < 0.05, (B) Genomic position of circSTAG2(16-25), which is generated by back splicing of exons 16-25.
Is Expressed at Higher Levels in Urine in Recurrent Samples