Abstract
Introduction
Bladder cancer (BC) is sensitive to radiation treatment and a subset of patients experience radiation-induced injuries including shrinkage of bladder due to bladder fibrosis.
Methods
This study is a retrospective cohort study. Three Japanese BC patients were randomly selected. Using a microRNA (miRNA) array, comparing their samples with or without radiation-induced injuries, we have checked the clustering of miRNA expression.
Results
Hsa-miR-130a, hsa-miR-200c, hsa-miR-141, and hsa-miR-96 were found to be highly expressed (>50 times) in patients with fibrotic bladder shrinkage (FBS) compared to those with intact bladder (IB) function. In patients with FBS, hsa-miR-6835, hsa-miR-4675, hsa-miR-371a, and hsa-miR-6885 were detected to have lesser than half expression to IB patients. We have analyzed the significance of these genes in relation to overall survival of 409 BC patients retrieved from the Cancer Genome Atlas data set. All available cutoff values between the lower and upper quartiles of expression are used for the selected genes, and false discovery rate using the Benjamini-Hochberg method is computed to correct for multiple hypothesis testing. We have run combined survival analysis of the mean expression of these four miRNAs highly expressed in FBS patients. 175 patients with high expression had a longer median survival of 98.47 months than 23.73 months in 233 patients with low expression (hazard ratio [HR]: 0.53; 0.39–0.72, log-rank p value: 7.3e−0.5). Combination analysis of all 8 genes including hsa-miR-6835, hsa-miR-4675, hsa-miR-371a, and hsa-miR-6885 showed the same HR for OS. Target scanning for these miRNAs matched specific cytokines known as an early biomarker to develop radiation-induced fibrosis.
Conclusions
BC patients with fibrotic radiation injury have specific miRNA expression profile targeting profibrotic cytokines and these miRNAs possibly render to favorable survival.
Keywords: Radiation, MicroRNA, Bladder cancer
Introduction
Bladder cancer (BC) is one of the most common malignancies, which is classified by the degree of bladder wall invasion. Treatment strategy for muscle-invasive bladder cancer (MIBC) that invades the muscularis propria totally differs from that of non-muscle-invasive bladder cancer (NMIBC), based on the metastatic potential in MIBC. Standard treatment for MIBC is radical cystectomy. An alternative to cystectomy is trimodality treatment, which is comprised of transurethral resection, external beam radiation therapy, and chemotherapy. External beam radiation therapy plays a crucial role in treating BC, especially when lymph nodes are involved. Pathological downstaging to NMIBC or pT0 at radical surgery is a favorable prognostic factor. However, lymph node metastases are still present in 4.1 and 4.6% of patients with a complete downstaging from cT2-4aN0M0 BC to pT0 or near complete downstaging to pTa/Tis/1 [1]. Radiation therapy also has limitations and complications for the urinary tract, including acute phase complications such as frequent urination and pain during urination or late complications such as chronic urinary frequency and difficulty urination, hematuria, and shrinkage of the fibrotic bladder. All these complications impair patients’ quality of life. MicroRNAs (miRNAs) play a significant role in the context of radiation therapy for BC. In the context of regulation of cellular response, miRNAs are posttranscriptional regulators that influence how cells respond to ionizing radiation-induced damage, where miRNAs modulate relevant genes involved in this process [2–4]. Experimental study has highlighted miRNA involvement in their potential to enhance radiotherapy effectiveness [5]. Hence, miRNAs can serve as predictive biomarkers, helping assess an individual’s response to radiotherapy [6–8]. Based on these backgrounds, we aimed to investigate the development of bladder fibrosis and the efficacy of radiation therapy in MIBC patients. The present study further compared the expression pattern of miRNAs between patients who developed complication due to bladder fibrosis after radiation therapy and those who did not and correlated the overall survival rate of patients who developed bladder fibrosis. The results of the present study may help urologists decide treatment strategies and individualization to minimize complications and improve treatment outcomes.
Methods
Validation of the Expression Profiles of miRNAs in Quantitative Real-Time Polymerase Chain Reaction
Three Japanese patients with advanced MIBC were enrolled in the present study. Patient 1 was a 73-year-old male patient with the tumor being diagnosed as T3 stage (control); patient 2 was a 70-year-old male – the tumor was T3 stage (severe bladder fibrosis); patient 3 was a 77-year-old male – the tumor was T2 stage as well (severe bladder fibrosis). Histopathology of the specimens was determined by transurethral resection of the bladder tumor. The histologic grade and tumor type of these 3 patients were high-grade urothelial carcinoma. The tumor stage of patients 1–3 was cT3N1M0 stage III, cT2N0M0 and stage III, cT2N0M0 and stage II, respectively, based on the American Joint Committee on Cancer (AJCC) TNM staging system. The tumor morphology of all 3 patients was sessile and multiple. Before the samples were taken, none of these 3 patients underwent chemotherapy and radiotherapy. All 3 patients underwent curative treatment. All 3 patients were cured with no evidence of disease. Two years after the end of the radiation therapy for the whole pelvis along with the bladder, cases 2 and 3 presented with urinary incontinence caused by the shrinkage of the bladder. Renal function remained unchanged. Extensive urinary investigation was performed, with normal urinalysis and negative viral and bacterial tests. Both patients refused bladder removal necessary with construction of an ileal conduit requiring the passage of an indwelling urinary catheter. The written informed consents of all the participants were obtained. Bladder specimens were immediately stored in the tissue in nitrogen after the resection. Whole RNA was extracted from the tissue by the 3D-Gene® RNA extraction reagent (Toray, Tokyo, Japan). Upon extraction from the tissue sample, the tissue was sliced into small pieces smaller than 5 mm. RNA was labeled and hybridized on the chip with 3D-Gene® miRNA labeling kit to detect >2,500 types of miRNAs [9–12]. Normalized data processing was conducted thereafter. The microarray was scanned, and the images were obtained. To generate raw data, the 3D-GeneH scanner 3000 (Toray, Tokyo, Japan) was used to extract the fluorescent signals from images. The expression status of miRNAs was normalized by the removal of the mean background signal intensity from the entire set of miRNAs in each microarray, which is generally called globally normalization method, with threshold for detected signal > the mean + 2 × SD of the blank spot signals. The microarray assay demonstrated that 701 miRNAs were expressed, in which 261 miRNAs were upregulated and 371 miRNAs were downregulated in patients 2; similarly, 132 miRNAs were upregulated and 113 miRNAs were downregulated in patients 3 according to p value (<0.05) and fold change (FC >2-fold). Previous studies have suggested that the altered expression of miRNAs was associated with poor prognosis of BC. To synthesize multiple microarray-based human BC miRNA expression profiling, we employed a vote-counting strategy to identify several consistent differentially expressed miRNAs, of which rank potential molecular markers widely adopted in the meta-analysis [13, 14]. We ranked miRNAs according to the importance of each miRNA. We considered the number of consistent comparisons reported previously, the total number of samples in agreement, and average fold changes reported for comparisons in agreement. For three of these, total sample size was deemed a more important factor than average fold change.
Validation of the Expression Profiles of miRNAs in the Cancer Genome Atlas Database
To assess multivariable prognostic miRNA expression profiles as biomarkers and to evaluate in several cohorts, curated and updated tool of miRNA expression levels associated with outcome evaluation that provides survival analysis established by Nagy et al. [15] was utilized. The system includes gene chips and RNA-seq data sources. The primary purpose of the tool is to identify and validate survival markers based on meta-analysis [15]. The distribution of tumor stage of 405 BC patients were stage I (5, 1.2%), stage II (130, 32.1%), stage III (138, 34.1%), and stage IV (132, 32.6%). High mutation burden status was detected in 204 patients (50.4%). Patient data were searched for keywords related to malignancies, survival information, and miRNA signatures [16]. Cohort searches are based on four large databases consists of GEO (http://www.ncbi.nlm.nih.gov/geo), GEOmetadb [17], ArrayExpress (https://www.ebi.ac.uk/arrayexpress/), and the Cancer Genome Atlas (https://tcga-data.nci.nih.gov/tcga). Platform-based analyses were conducted as reported by Lanczky et al. [18] that interactivity is increased by the usage of JavaScript and Ajax technologies. The server is running on Debian Linux (www.debian.org) and is powered by Apache (www.apache.org). In precise, MiRNAs were input as a gene symbol, with the truncation value set as the median expression value, and BC was selected for analysis in the histology column. The Kaplan-Meier survival curves were plotted, and the number at-risk is indicated below the main plot. The survival analysis results of BC patients were represented by hazard ratio (HR), 95% confidence intervals (CI), and log-rank p values calculated. Proportional hazard was computed by the COX analysis. Statistical significance was set at p < 0.05. Bonferroni correction was executed for studies while simultaneously publishing.
TargetScan8.0 was utilized to predict miRNA targets by looking for the presence of conserved 8mer, 7mer, and 6mer sites that match the seed region of every miRNA, then to rank miRNA based on the predicted efficacy of targeting, calculated using cumulative scores considering all the features of the sites (https://www.targetscan.org). The obtained results were checked with MIRZA-G (http://www.clipz.unibas.ch/index.php?r=tools/sub/mirza_g) that is using the same strategies to TargetScan8.0, including sequence features, thermo-stability, evolutionary conservation, and site accessibility.
Results
To find a panel of miRNAs as biomarker for fibrotic bladder shrink (FBS) caused by X-ray radiation, the present study initiated with the selection of specific miRNA candidates based on the comparison of expression levels in tissue between BC patients with bladder fibrillation shrinkage and intact bladder (IB) adjusted by representative BC using the 3D-Gene miRNA array. Selection of candidates BC miRNA for miRNA panel was done from the comprehensive miRNA array-based approach. We selected candidate miRNAs for FBS detection based on comparison of the expression levels of each miRNA between BC patients with FBS and IB counterpart (shown in Fig. 1). To implement a clustering algorithm, Pearson correlation was used to measure similarity (shown in Fig. 1). Fold change was used in analysis of expression data in microarray and was calculated as the ratio of the difference. Value was the log 2 transformation of the normalized ratio of the average red signal and the average green signal, and genes that have 2 times larger log 2 of red/green normalized ratio were used. FBS compared with IB samples were assessed. In FBS, significantly higher miRNA expressions of hsa-miR-130a, hsa-miR-200c, hsa-miR-141, hsa-miR-96 were found with a fold change >50 (shown in Fig. 2). The expressions of hsa-miR-6835, hsa-miR-4675, hsa-miR-371a, and hsa-miR-6885 in IB were found with a fold change of >2 compared to FBS (shown in Fig. 2).
Fig. 1.
Clustering and heat maps by two different BC types. Each row in the diagram represents a gene and each column a tumor sample. The color saturation represents differences in gene expression across the samples. Red signals indicate expression higher than the median, and green signals indicate expression lower than the median. The color intensity indicates degree of gene regulation.
Fig. 2.
Heat maps by two different BC types. Variable ratio <2 times or >50 times had 4 genes for each.
To explore the differential expression of mRNAs between FBS and IB, we utilized the interface analyses as reported by Lanczky et al. [18] which was set up to enable reproduction of computations in a platform-independent user interface. Spearman correlation of hsa-miR130a, hsa-miR-200c, hsa-miR-141 to hsa-miR-96 were 0.1984 (p = 1e−04), 0.619 (p < 1E−04), 0.6703 (p < 1E−04). We have run combined survival analysis of mean expression of these four miRNAs. 175 patients with high expression had longer median survival of 98.47 months than 23.73 months in 233 patients with low expression (HR: 0.53; 0.39–0.72, log-rank p value: 7.3e−0.5). Combination analysis of all 8 genes including hsa-miR-6835, hsa-miR-4675, hsa-miR-371a, and hsa-miR-6885 rendered the same HR for OS (shown in Fig. 3).
Fig. 3.
The prognostic value of a set of miRNAs in patients with BC was tested by a Kaplan-Meier estimate of the duration of overall survival in patients.
Prediction of biological targets of miRNAs were conducted searching for the presence of conserved 8mer, 7mer, and 6mer sites that match the seed region of each miRNA using TargetScan8.0 (https://www.targetscan.org). Bladder shrinkage and fibrosis are reported to occur via transforming growth factor (TGF)-β pathway [19–21]. We investigated whole TGF-β fibrosis pathway-related proteins in relation to eight miRNAs (Tables 1, 2). Transforming growth factor beta regulator 1, transforming growth factor beta receptor associated protein 1, transforming growth factor beta receptor III, latent transforming growth factor beta binding protein 3, transforming growth factor beta 2, transforming growth factor, beta receptor II (70/80 kDa), transforming growth factor beta receptor 1 are all found to be the target of hsa-miR-6885, which was most intensively upregulated in IB patients. These seven TGF-β fibrosis pathway-related proteins were observed as target for four miRNAs found in IB patients (18/28, 64.2%), showing markedly high positivity compared to low positive target ratio observed in the four miRNAs found in FBS patients (6/28, 21.4%, Table 1). Another fibrosis-related UV radiation resistance-associated gene was focused [22, 23]. UV radiation resistance-associated gene was a target of hsa-miR-4675, hsa-miR-371a, and hsa-miR-6885 (Table 2).
Table 1.
TGF beta signaling pathway genes and relation to microRNAs
| Gene name | Transforming growth factor beta regulator 1 | Transforming growth factor, beta receptor-associated protein 1 | Transforming growth factor, beta receptor III | Latent transforming growth factor beta-binding protein3 | Transforming growth factor, beta 2 | Transforming growth factor, beta receptor II (70/80 kDa) | Transforming growth factor, beta receptor 1 |
|---|---|---|---|---|---|---|---|
| Target gene | TBRG1 | TGFBRAP1 | TGFBR3 | LTBP3 | TGFB2 | TGFBR2 | TGFBR1 |
| hsa-miR-130a | – | Target | – | – | Target | Target | Target |
| hsa-miR-200c | – | – | – | – | – | – | – |
| hsa-miR-141 | – | – | – | – | Target | – | – |
| hsa-miR-96 | – | – | – | – | – | – | Target |
| hsa-miR-6835 | – | Target | Target | – | – | Target | – |
| hsa-miR-4675 | Target | Target | Target | Target | Target | Target | Target |
| hsa-miR-371a | – | – | – | – | Target | – | – |
| hsa-miR-6885 | Target | Target | Target | Target | Target | Target | Target |
Table 2.
UV radiation and X-ray radiation resistance-associated protein and relation to microRNAs
| Gene name | UV radiation resistance associated | X-ray radiation resistance associated 1 |
|---|---|---|
| Target gene | UVRAG | XRRA1 |
| hsa-miR-130a | – | – |
| hsa-miR-200c | – | – |
| hsa-miR-141 | – | – |
| hsa-miR-96 | – | – |
| hsa-miR-6835 | – | – |
| hsa-miR-4675 | Target | Target |
| hsa-miR-371a | Target | – |
| hsa-miR-6885 | Target | Target |
UVRAG, UV radiation resistance-associated gene.
Discussion
The cytotoxic effects of ionizing radiation are employed in cancer radiotherapy, but its actions also influence cellular signaling pathways. Recent publications have investigated molecular changes that drive radiation-induced fibrosis in cancer survivors using patients’ samples with history of radiation therapy [24–28]. In cancer survivors with history of radiation therapy, elevated levels of varying growth factors including TIMP1, TIMP2, PAI 1, VEGF-A, HGF, and TGF-β are detected, suggesting that fibrosis and vascular damage drive the development of radiation-induced cystitis [24]. Oxidative stress-induced activation of TGF-β with activation of SMAD signaling, TNF-α elaboration, and activation of the angiotensin converting enzyme mediated production of angiotensin II with resulting activation of profibrotic cytokine signaling and vasoconstriction [27]. Among them, TGF-β is considered a key profibrotic cytokine for the initiation, development, and persistence of radiation fibrosis [25, 27, 28]. Radiation-induced fibrosis is a clinically significant intermediate-to-late-occurring side effect of radiotherapy. Hosseini et al. [25] aimed to compare TGF-β1 serum levels by enzyme-linked immunosorbent assay technique in early-stage breast cancer patients treated with whole-breast radiation therapy plus boost versus accelerated partial breast irradiation. Three months after radiotherapy, significantly increased serum TGF-β1 level was observed in the radiation therapy group, suggesting that onset of TGF-β1 takes time [25]. Wang et al. [29] investigated lung tissue injury in a mouse radiation model and found the lung injury scores and pulmonary fibrosis indices in the irradiation group were higher than those in the control group. Lung pneumonia score, pulmonary fibrosis index, expression of TGF-β1, and p-Smad3 in the lung tissue of mice in irradiation were higher than control [29].
Radiotherapy effectively treats pelvic malignancies in the pelvis including BC, prostate cancer, and rectal cancer but is associated with mid-to-late-phase side effects such as cystitis and proctitis in some patients. Furthermore, it could cause mucosal cell death, inflammation, hematuria, and bladder fibrosis [30]. Bladder cavity is reduced by radiation cystitis, which leads to dysuria and incontinence. Radiation-induced bladder neck strictures present more often with lower urinary tract symptoms as a side effect of their prior radiotherapy treatment. Crawford et al. [31] reported that radiation-induced side effects in the lower urinary tract that result in lower urinary tract symptoms include injury to peripheral neurons, interstitial fibrosis of the bladder, and loss of muscle fibers in the muscularis propria, indicating the lower urinary tract can suffer organ shrinkage by fibrotic changers [31].
In our study, we have come up with the insight that fibrotic changes in the radiated bladder occur via alteration of specific miRNAs that negatively regulate specific key cytokines known as a biomarker to develop radiation-induced fibrosis. The ratio of noncoding to total genomic DNA increases with biological complexity [32, 33], driven by enhanced information content. Previously deemed unnecessary, introns and genomic DNA may have functional roles. Only 2% of the mammalian genome encodes miRNAs, which could play a role in bladder cell transformation. Previous study has showed that long noncoding RNAs (lncRNAs) H19 are implicated in regulating epithelial-to-mesenchymal transition of BC cells and the reverse mesenchymal-to-epithelial transition [34]. We have found patients with bladder fibrosis lacked the presence of hsa-miR-6835, hsa-miR-4675, hsa-miR-371a, and hsa-miR-6885; all of these are negative regulators for TBRG1, TGFBRAP1, TGFBR3, LTBP3, TGFB2, TGFBR1, and TGFBR2.
We hope our findings help improve the effectiveness of radiotherapy in the treatment of BC including the most appropriate patient selection and the best radiation prescription dose for treating BC, potentially improving cure rates and reducing adverse effects. This study did have some limitations.
First, this study included 3 Japanese BC patients; this is a relatively small sample size. Therefore, it may be limitation to generalize the conclusion in this study.
Second, it is about confounding factors. We selected randomly 3 BC patients who after radiation therapy. They had almost the same degree of TNM stage, and no one received chemotherapy and radiotherapy at the time of sample collection. However, they did not investigate to what degree they had dysuria before radiotherapy. This may affect the outcome not a little.
Conclusion
Radiotherapy for BC could cause chronic fibrosis, resulting in bladder shrinkage. Specific miRNAs strongly discriminated healthy controls from patients with bladder fibrosis.
Acknowledgment
We are grateful to the staff members of the Department of Urology, Osaka Medical and Pharmaceutical University, for their suggestions and assistance.
Statement of Ethics
The present study was approved by the Institutional Review Board of the Principal Hospital of Osaka Medical and Pharmaceutical University. (approval number: RIN−750−2571, 2022) Written informed consent was obtained from each participant in this study.
Conflict of Interest Statement
The authors have no conflicts of interest to disclose.
Funding Sources
This study was not supported by any sponsor or funder.
Author Contributions
Ko Nakamura, Takaya Ohno, Teruo Inamoto, Tomoaki Takai, Taizo Uchimoto, Tatsuo Fukushima, Kazuki Nishimura, Yusuke Yano, Kyosuke Nishio, Shoko Kinoshita, Tomohisa Matsunaga, Keita Nakamori, Takeshi Tsutsumi, Takuya Tsujino, Hirofumi Uehara, Kazumasa Komura, Kiyoshi Takahara, and Haruhito Azuma contributed to this work. Ko Nakamura and Takaya Ohno acquitted the data. Ko Nakamura, Takaya Ohno, and Teruo Inamoto analyzed and interpreted the data. Ko Nakamura, Takaya Ohno, and Teruo Inamoto wrote the manuscript.
Funding Statement
This study was not supported by any sponsor or funder.
Data Availability Statement
All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.
Supplementary Material
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.
