Circ_0047339 promotes the activation of fibroblasts and affects the development of urethral stricture by targeting the miR-4691-5p/TSP-1 axis

Urethral stricture is related to scar tissue fibrosis, but its pathogenesis is still unclear. This study aims to explore the regulatory mechanism of circular RNA (circRNA) in the occurrence and development of urethral stricture. CircRNA microarray was employed to analyze circRNA expression profiles between human urethral scar tissue and normal urethral tissue. The results of circRNA microarray showed that there were 296 differentially expressed genes between urethral scar tissue and normal urethral tissue. The enrichment analysis of Kyoto encyclopedia of genes and genomes showed that these circRNAs were significantly correlated with ECM–receptor interaction. The first nine differentially expressed circRNA were selected to predict the circRNA–miRNA network. RT-qPCR results showed that circ_0047339 was upregulated considerably in urethral scar tissue. Urethral scar fibroblasts were isolated from human urethral scar tissue and cultured in vitro. After silencing circ_0047339, the proliferation of urethral scar cells decreased significantly, and the expressions of Collagen I (COL-1) and α-smooth muscle actin (α-SMA) also reduced. As a competing endogenous RNA, circ_0047339 could increase the expression of TSP-1 by competitively binding miR-4691-5p. In addition, miR-4691-5p mimic transfection could inhibit the proliferation of urethral scar fibroblasts and the presentation of thrombospondin-1 (TSP-1), α-SMA and COL-1, while circ_0047339 overexpression eliminated this inhibition. Our results showed that circ_0047339 might promote the growth and fibrosis of urethral scar fibroblasts through miR-4691-5p/TSP-1 axis, thus promoting the development of urethral stricture.


Materials and methods
Source of clinical specimens. From Jan. 2021 to Jun. 2021, six pairs of urethral scar and normal urethral tissue were collected in Xiangya Hospital. Patients' baseline information was summarized in Table 1. No matter it is trauma or iatrogenic stenosis, all patients with urethral stenosis underwent surgery at 2 months after the onset of the cause. The surgical specimens were free of local infection and ulcer, and no drug or radiation therapy was given before surgical resection. All specimens were obtained with the consent of patients and their families before operation and confirmed by pathological examination. The procedure used in this research followed the tenets of the Declaration of Helsinki and was approved by the Medical Ethics Committee of Xiangya Hospital Central South University.
CircRNA microarray analysis. The circRNA expression profile of the urethral scar and normal urethral tissue was detected using CapitalBio Technology Human CircRNA Array v2 (CapitalBio Technology, China). The total RNA of three urethral scar tissue and three normal urethral tissue were extracted. The purity and concentration of the RNA were determined by a NanoDrop ND-1000 instrument (Thermo Scientific, USA). The extracted RNAs were amplified and had reverse transcription into cDNA, then labeled with Cy3-dCTP. After purification, the labeled DNAs were hybridized into a microarray (CapitalBio Technology Human CircRNA Array v2). The circRNAs expression difference and statistical significance P value were calculated by GeneSpring GX software. Cluster analysis and graphical display were performed with Cluster3.0 software. The differential comparison is conducted to obtain differential genes according to the grouping information. Kyoto encyclopedia of genes and genomes (KEGG) Pathway analysis was performed on the linear mRNA transcripts corresponding to different circRNA 25 . The miRNA that circRNA might bind to was predicted by bioinformatics.
Primary cell extraction and culture. The urethral scar tissue or normal urethral tissue was repeatedly rinsed in PBS containing antibiotics to remove redundant epithelial tissue. The tissues were divided into small pieces of 5 mm × 5 mm in size. Then, we dropped the culture medium onto the tissue to keep it moist. The tissues were put into a centrifuge tube. We added 0.25% trypsin digestive juice-EDTA and collagenase, digested it in a constant temperature water bath at 37 °C for 4-5 h, and shake it once every hour. We filtered the suspension with a filter screen, took the filtered suspension for centrifugation, discard the supernatant, added 6 mL of fresh culture solution, and cultured it in a cell incubator at 37 °C and 5% CO 2 . The next day, most of the fibroblasts adhered to the wall, the cell culture medium was changed, and the cells were changed every 2-3 days. Finally, urethral scar fibroblasts and normal urethral scar fibroblasts were successfully separated and cultured. The α-smooth muscle actin (α-SMA) was selected as a marker of fibroblasts for detection.
Cell transfection. The small interference RNA (siRNA) specifically targeting circ_0047339 (si-circ_0047339), Lentivirus harboring circ_0047339 (LV-circ_0047339), miR-4691-5p mimic, and their corre-    Cell counting kit-8 (CCK-8) assay. According to the manufacturer's instructions, the CCK-8 kit (NU679, DOJINDO) was used to detect cell viability. The cells were inoculated into a 96-well plate (1 × 10 4 /well) and attached to the wall in a 5% CO2 incubator at 37 °C for 24 h. According to the demand, the cells were divided into different groups. Each group was provided with 6 compound holes 24 h after taking out the 96-well plate. We observed it under a microscope, added CCK-8 reagent and incubated it for 2 h. A microplate analyzer was utilized to detect the absorbance (OD) value at 450 nm, and the results were recorded.

5-Ethynyl-2′-deoxyuridine (EDU) assay.
According to the manufacturer's instructions, cell proliferation was performed by the EDU assay kit (Guangzhou RiboBio). The cells were inoculated into 96-well plates (1 × 10 4 /well) and incubated with 4% paraformaldehyde at room temperature for 30 min. Then, the cells were treated with 100 μL of 1 × Apollo ® staining reaction solution for 30 min. Next, 100 μL Hoechst 33342 reaction solution was added to each well and incubated for 30 min. Immediately after dyeing, we used a microscope (DSZ2000X, Beijing Cnmicro instrumentco., Ltd) to observe and take pictures.
Statistics. Statistical analyses were conducted using GraphPad Prism 9 software (GraphPad Software, Inc., USA). Each experiment was conducted at least three times. Statistical analysis among more than two groups and between two groups was performed using ANOVA and Student's t test. Data are presented as means ± standard deviations. P < 0.05 was considered statistically significant, P < 0.01 was considered a significant statistical difference, and P < 0.001 was considered an extremely significant statistical difference.
Ethical approval. The procedure used in this research followed the tenets of the Declaration of Helsinki and was approved by the Medical Ethics Committee of Xiangya Hospital Central South University (202112614). All specimens were obtained with the informed consent of patients and their families before operation and confirmed by pathological examination.

Results
CircRNA expression profile analysis of urethral scar tissue. We performed circRNA microarray to identify the differential circRNA expression between human urethral scar tissue and normal urethral mucosa tissue. Then, we determined the characteristics of circRNA in the urethral scar. Principal Component Analysis (PCA) was a statistical method to reflect the similarity of samples. By dimensionality reduction of data, the expression of samples was displayed in three-dimensional space. The result showed that the contribution rate of the top 3 principal components is 77.52% (Fig. 1A). The scatter plot showed the difference in circRNA expression between samples in the urethral scar group (case) and the normal urethral mucosa tissue group (control). Among them, those marked in red were up-regulated circRNA, those marked in green were down-regulated circRNA, and those marked in black were circRNA with no significant difference (Fig. 1B). In addition, we drew a volcano map together with P-value and fold change (FC), which were obtained by difference analysis. Based on the threshold of FC ≥ 2 and P-value ≤ 0.05, 296 differential circRNA were identified (Fig. 1C). Among them, 166 circRNA expressions were down-regulated, and 130 circRNA expressions were upregulated in human urethral scar tissue, compared with normal urethral tissue.
Interaction between circRNA and miRNA in urethral scar tissue. We next aimed to explore the functional changes caused by circRNA changes in patients with urethral fibrosis. Based on the microarray analysis data, the KEGG pathway of different circRNA groups was annotated and enriched. KEGG pathway statistical chart showed the first 30 enriched signal pathways ( Fig. 2A). The differential circRNA was significantly related to ECM-receptor interaction, Arrhythmogenic right ventricular cardiomyopathy (ARVC), Thyroid hormone synthesis, and Proteoglycans in cancer and Focal adhesion. CircRNA could combine with miRNA in a targeted way and indirectly regulate the translation of mRNA 27 . The miRanda software was used to select the differentially expressed circRNA to predict the target miRNA, and the circRNA-miRNA network diagram was drawn. The results showed the interaction between the first ten most significantly expressed circRNA and its target miRNA (Fig. 2B). www.nature.com/scientificreports/ The expression of circ_0047339/miRNA/TSP-1 interaction in urethral scar tissue. We collected human normal urethral and human urethral scar tissue, and determined the expressions of COL-1 and α-SMA via western blot assay. The expressions of COL-1 and α-SMA were significantly upregulated in human urethral scar tissue compared with normal urethral tissue (Fig. 3A). Next, we verified the expression of the first nine differential circRNA in the microarray analysis results by RT-qPCR. The results showed that circ_0047339 was upregulated in human urethral scar tissue, and the most significant difference (Fig. 3B). Therefore, we choose circ_0047339 for follow-up research. The above results showed that ECM-receptor interaction was the most significant KEGG pathway for differential circRNA enrichment. TSP-1 is a matricellular protein in the ECM, which mediates cell-matrix and cell-cell interactions 14,28 . We speculated that TSP-1 might be an important target gene downstream of circ_0047339. Therefore, the expression of TSP-1 was detected by RT-qPCR in urethral scar tissue. TSP-1 expression was upregulated in urethral scar tissue compared to normal urethral tissue (Fig. 3C).
Circ_0047339 regulated the fibrosis of urethral fibroblasts. Next, to study the function of circ_0047339, we extracted human primary urethral fibroblasts and human primary urethral scar fibroblasts. IF detection showed that the expression of COL-1 and α-SMA in human primary urethral scar fibroblasts was higher than that in human primary urethral fibroblasts (Fig. 4A). Western blot results were consistent with the IF results (Fig. 4B). In addition, the protein level of TSP-1 increased in human primary urethral scar fibroblasts (Fig. 4B). The results of RT-qPCR showed that circ_0047339 and TSP-1 were upregulated in human primary urethral scar fibroblasts, while miR-4691-5p was the opposite (Fig. 4C). The expression of circ_0047339 in human primary urethral scar fibroblasts was silenced by transfection of si-circ_0047339 (Fig. 4D). Meanwhile, the expression of miR-4691-5p in the si-circ_0047339 group was upregulated than in the si-NC group (Fig. 4D).
Western blot results showed that the protein levels of TSP-1, COL-1 and α-SMA were down-regulated in si-circ_0047339 group compared to si-NC group (Fig. 4E). The results of IF were consistent with those of western blot (Fig. 4F). Compared with the si-NC group, the cell viability of si-circ_0047339 group decreased (Fig. 4G).
The EDU test results also showed cell proliferation in the si-circ_0047339 group decreased (Fig. 4H). These results suggested that silencing si-circ_0047339 could reduce the vitality of urethral fibroblasts, inhibit proliferation and alleviate fibrosis.
Circ_0047339 acts as a ceRNA in TSP-1 regulation by sponging miR-4691-5p. The targeting relationship between circ_0047339 and miR-4691-5p was analyzed by bioinformatics. Figure 5A showed the targeted binding sites of circ_0047339 and miR-4691-5p. Dual-luciferase assay results showed that miR-4691-5p mimic transfection significantly reduced the luciferase activity of circ_0047339-WT, but did not reduce the luciferase activity of circ_0047339-MUT group (Fig. 5B). TSP-1 3′UTR contained the potential binding site of miR-4691-5p (Fig. 5C). Then the luciferase reporter plasmid was constructed, which includes 3′UTR regions of TSP-1 mRNA for luciferase detection. The results showed that miR-4691-5p mimic significantly inhibited the www.nature.com/scientificreports/ luciferase activity of TSP-1-WT, but had no effect on the TSP-1-MUT group (Fig. 5D). The expression level of TSP-1 mRNA in human primary urethral scar fibroblasts transfected with miR-4691-5p mimic was detected by RT-qPCR. The results showed that TSP-1 mRNA in the miR-4691-5p mimic group was significantly downregulated compared with the mimic-NC group (Fig. 5E). These results indicated that circ_0047339 could be used as a molecular sponge of miR-4691-5p to regulate the expression of TSP-1.

Circ_0047339/miR-4691-5p/TSP-1 interaction network was involved in regulating the proliferation, ECM deposition and collagen synthesis of urethral scar fibroblasts.
Because circ_0047339 sponged miR-4691-5p, we subsequently determined the role of miR-4691-5p in vitro. MiR-4691-5p mimic transfection could inhibit the expression of α-SMA and COL-1 protein in urethral scar fibroblasts, while circ_0047339 overexpression could eliminate this inhibition (Fig. 6A,B). These results indicated that circ_0047339/miR-4691-5p interaction regulated ECM deposition and collagen synthesis in urethral scar fibroblasts. As we have proved that TSP-1 is the target of miR-4691-5p, we detected the expression of TSP-1 mRNA and protein by RT-qPCR and western blot. MiR-4691-5p mimic transfection could inhibit the expression of TSP-1 in urethral scar fibroblasts, while circ_0047339 overexpression could eliminate this inhibition (Fig. 6B,C). In addition, we found that miR-4691-5p mimic transfection could inhibit the vitality and proliferation of urethral scar fibroblasts. Overexpression of circ_0047339 could reverse these results (Fig. 6D,E). These results indicated that circ_0047339/miR-4691-5P/TSP-1 network was involved in the regulation of proliferation, ECM deposition and collagen synthesis of urethral scar fibroblasts.

Discussion
In this study, based on the expression profile analysis of circRNA, we identified circ_0047339 as significantly upregulated circRNA in urethral scar tissue. The loss of function experiment showed that circ_0047339 was related to cell viability, proliferation and fibrosis. Circ_0047339 played its role as a ceRNA that competitively bound miR-4691-5p and then eliminated the endogenous inhibition of miR-4691-5p on the target gene TSP-1. Thus, it could promote the expression of COL-1 and α-SMA and then cell proliferation and fibrosis progress. www.nature.com/scientificreports/ These results revealed that circ_0047339 promoted the growth and fibrosis of urethral scar fibroblasts through the ceRNA mechanism. At present, there are few research articles on the relationship between circRNA and urethral stricture. The existing literature mainly focuses on renal fibrosis 20,27 . Other studies have found that circRNA is related to the mechanism of keloid formation [29][30][31] . Our study used gene chip technology to analyze the differentially expressed circRNA in urethral scar tissue. We found that compared with the normal control group, there were 130 upregulated circRNA and 166 down-regulated circRNA in urethral scar tissue. In addition, KEGG pathway analysis found that differentially expressed circRNA was significantly correlated with ECM-receptor interaction. This opens the door for studying circRNA and urethral stricture, but the relationship between circRNA and the development mechanism of urethral stricture needs further research.
In order to explore how differential circRNA participates in the occurrence and development of urethral stricture, we verified its expression in urethral scar tissue. The results showed that the expression of circ_0047339 in human urethral scar tissue was upregulated, and the difference was the most significant. Therefore, we chose circ_0047339 for the follow-up experiment. Urethral scar formation caused by the overactivation of urethral fibroblasts is the core cytobiological event of urethral stricture 32,33 . We selected urethral fibroblasts to verify the function of circ_0047339. The results showed that circ_0047339 was significantly overexpressed in urethral scar fibroblasts compared with normal urethral fibroblasts. By silencing the expression of circ_0047339, we found that the cell viability, proliferation, and expression of α-SMA and COL-1 of urethral scar fibroblasts decreased. This was consistent with previous results 34 . This evidence suggested that circ_0047339 might participate in urethral stricture by influencing the growth of urethral scar fibroblasts, ECM deposition, and collagen synthesis. It was recommended that circ_0047339 might play an important role in the occurrence and development of urethral stricture.
The research on the biological function mechanism of circRNA has made rapid progress. Among them, circRNA has received the most attention in the function of the miRNA sponge 35,36 . For example, circPTPN12 promotes keloid fibroblasts' growth by activating the Wnt pathway by sponging miR-21-5p 37 . Through dualluciferase reporter gene detection, we found the interaction of circ_0047339 of miR-4691-5p. MiR-4691-5p can promote the development of liver cancer 38 , but it has not been studied in urethral stricture. Our study was the first to explore the role of miR-4691-5p in urethral stricture. This study found that miR-4691-5p was down-regulated in urethral scar tissue and fibroblasts compared with the control group. Moreover, miR-4691-5p mimic transfection could inhibit the vitality and proliferation of urethral scar fibroblasts. It also could inhibit the expression of α-SMA and COL-1. We speculated that miR-4691-5p might limit fibrogenic signaling in urethral strictures. The dual-luciferase reporter assay showed that the translation activity of TSP-1 3′UTR was significantly inhibited www.nature.com/scientificreports/ by miR-4691-5p mimic. Correlation analysis showed that miR-4691-5p expression was negatively correlated with the expression of circ_0047339 and TSP-1 in urethral scar tissue. In urethral scar fibroblasts, inhibition of circ_0047339 could up-regulate miR-4691-5p expression and inhibit the expression of TSP-1. These data suggested that circ_0047339 could be used as a molecular sponge of miR-4691-5p to regulate the expression of TSP-1. Inhibition of TSP-1 expression has been proved to inhibit hypertrophic scar development 39,40 . TSP-1 is a regulatory factor that promotes the process of fibrosis, and TGF-β1 can be activated by TSP-1, thus promoting ECM deposition and collagen synthesis 41,42 . Clinical studies have revealed that TGF-β1 levels are significantly up-regulated in patients with urethral strictures 43 . TGF-β1 signaling-mediated hyperactivation of urethral fibroblasts contributes to the progression of traumatic urethral strictures 44 . Targeted intervention in the TSP-1/TGF-β pathway is a therapeutic approach to preventing fibrotic diseases 16 . Our results demonstrated that transfection of miR-4691-5p mimic could inhibit the expression of TSP-1 in urethral scar fibroblasts, thereby inhibiting fibrosis, while overexpression of circ_0047339 could reverse its inhibitory effect. These results suggested that circ_0047339/miR-4691-5p/TSP-1 network participates in the growth and activation of urethral scar fibroblasts. It is important to emphasize some limitations related to this study. First, the role of only circ_0047339 was verified, while the expression profile of other circRNAs in urethral stricture remains to be explored. In addition, circ_0047339 was highly expressed in urethral scar tissue, but the reason or mechanism for its high expression needs to be further studied through systematic experimental analysis. Finally, TSP-1 is an extracellular matrix protein that mediates cell-matrix and cell-cell interactions 14 . Many studies have demonstrated that TSP-1 is the main regulator of TGF-β activation and can regulate the expression of TGF-β/Col-1/α-SMA 16,41,45 . Herein, we found that circ_0047339 promoted the expression of TSP-1 by sponging miR-4691-5p in urethral stricture. Meanwhile, down-regulation of circ_0047339 inhibited the expression of Col-1/α-SMA. Therefore, we speculated that circ_0047339 might affect Col-1/α-SMA by affecting the miR-4691-5p/TSP-1 axis. However, the mechanism of TSP-1's effect on Col-1/α-SMA is quite complex. More experimental data will be needed to prove its specific regulatory mechanism. This is the limitation of our work. We will conduct cell and animal experiments to analyze this in-depth in future research.

Conclusion
Our research showed that circRNA could regulate the development of urethral stricture. Circ_0047339 was upregulated in human urethral scar tissue and urethral scar fibroblasts. Circ_0047339 silence could inhibit the proliferation of urethral scar fibroblasts and the expression of α-SMA and COL-1. Circ_0047339 regulated fibroblast proliferation, ECM deposition and collagen synthesis by increasing TSP-1 expression as an endogenous miR-4691-5p sponge.

Data availability
All data included in this study are available upon request by contact with the first author or corresponding author. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.