Abstract
Objective: To analyze the expression profile variation of lncRNAs in normal urinary bladder tissue and urothelial carcinoma of the urinary bladder through microarray technology. The differentially expressed lncRNAs were identified and classified, and their biological information was analyzed. The data obtained in the study will prove helpful for the diagnosis, treatment, and prevention of urothelial carcinoma of the bladder. Materials and methods: Three specimens of urothelial carcinoma of the bladder and three specimens of normal bladder tissue were identified by histology. The total RNA was isolated from the bladder urothelial carcinomas and normal tissue and purified. The targets were mixed and hybridized with the genes on the microarray, which contained thirty thousand lncRNAs. The bladder urothelial carcinomas were then compared with the normal bladder tissue. The lncRNAs that were differentially expressed between the two groups were identified based on the signal-to-noise ratios using the Agilent Feature Extraction software and were analyzed with the Agilent Genespring GX software (Agilent). The outcome was obtained, and the biological information of these genes was deposited in GenBank. Results: The expression profile of lncRNAs was significantly different between normal bladder tissue and urothelial carcinoma of the bladder. Compared with normal bladder tissue, 1,122 lncRNAs exhibited at least a twofold, significant difference (P < 0.05) and are thus regarded as differentially expressed lncRNAs. Of these, 734 and 388 lncRNAs were upregulated and down regulated. The differentially expressed lncRNAs in the urothelial carcinoma of the bladder are distributed on every chromosome, and most of these lncRNAs are distributed on chromosomes 1, 2, 3, 4, 6, and X. Conclusions: Urothelial carcinoma of the bladder is a complicated disease that involves the regulation of multiple genes and the participation of multiple chromosomes. Some of the differentially expressed lncRNAs that were upregulated, such as AK124776, lincRNA-RAB12-1, KRT8P25, RP11-474J18.4, AC000110.1, KRT8P13, KRT8P10, BC072678, and downregulated, such as nc-HOXB9-206, RP11-160A10.2, nc-HOXA11-86, nc-HOXD10-7, nc-HOXB9-205, CES4, nc-HOXD12-3, systematic research on these lncRNAs will help clarify the mechanisms of urothelial carcinoma of the bladder and guide the early diagnosis and treatment of this cancer in the future.
Keywords: lncRNA, gene microarray, urinary bladder, urothelial carcinoma, expression profile
Introduction
Carcinoma of the bladder is the ninth most common malignancy of the genitourinary tract [1] and has several pathological types, including urothelial cell carcinoma (UCC), adenocarcinoma, and squamous cell carcinoma. UCC is the most common pathological type of bladder carcinoma. Early diagnosis and early treatment is important for the cure of UCC. The objective of our research is to identify a suitable tumor marker with high sensibility and high specificity to allow the early detection of UCC. It is well known that the pathogenesis of UCC is complicated and is the result of the interaction of multiple factors, including heredity, environment, and metabolism factors. Among these, smoking remains a major risk factor [2,3]. The germination of carcinoma is related to multiple cytokines and proteins that are regulated by many types of genes. In recent years, we have started to determine the cellular- and molecular-level changes induced by UCC and have attempted to investigate the cellular and molecular mechanism to ultimately find a UCC-specific marker. To date, we have obtained a large number of valuable experimental evidence. However, it is difficult to explain the germination of UCC through a single molecule or gene. The procedure involves multiple processes and multiplegenes. With the development of the combination of genomic and proteomic studies, recent studies are limited principally to DNA, coding RNA, and protein. As a result, an increasing number of markers that are likely related to UCC have been found and reported [4-18]: (1) DNA methylation markers (e.g., NMP22), (2) mRNA markers (e.g., survivin, PSCA, p63, and c-FLIP), and (3) cytokine or protein markers (e.g., BLCA-1, BLCA-4, DJ-1, HSP90a, CCL 18, OCT4, COX-2, IL-8, and HER-2). Nevertheless, we have not found a specific marker for UCC. With the development of sensitive microarrays, numerous non-coding RNAs (ncRNA) have been found, and this number far exceeds the number of coding RNAs in the mammalian transcriptome. Increasing studies have revealed that long non-coding RNAs (lncRNA) have a variety of important functions, including roles in transcription, splicing, translation, nuclear factor trafficking, imprinting, genome rearrangement, and chromatin modification [19-21]. Thus, we hypothesize that lncRNAs, in combination with mRNAs, are also involved in the germination and development of tumor. The aim of this study is to analyze the expression profile variation of lncRNAs in normal urinary bladder tissue and urothelial carcinoma of the bladder through microarray technology to explore the function of lncRNAs in UCC and to attempt to identify a specific gene that will prove helpful to the diagnosis, treatment, and prevention of UCC.
Materials and methods
Samples
The RNA quantity and quality were measured using a NanoDrop ND-1000, and the RNA integrity was assessed by standard denaturing agarose gel electrophoresis.
Microarray
The Arraystar Human LncRNA Array v2.0 was designed for researchers who are interested in profiling both LncRNAs and protein-coding RNAs in the human genome. A total of 33,045 LncRNAs were collected from authoritative data sources, including RefSeq, UCSC Known Genes, Ensembl, and the related literature.
RNA labeling and array hybridization
The sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, the mRNA was purified from the total RNA after the removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method. The labeled cRNAs were purified with a RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured using a NanoDrop ND-1000. A mass of 1 μg of each labeled cRNA was fragmented through the addition of 11 μl of 10X blocking agent and 2.2 μl of 25X fragmentation buffer. The mixture was then heated at 60°C for 30 min. Then, 55 μl of 2X GE hybridization buffer was added to dilute the labeled cRNA, and 100 μl of the hybridization solution was dispensed into the gasket slide and assembled on the lncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent hybridization oven. The hybridized arrays were washed, fixed, and scanned using an Agilent DNA microarray scanner (part number G2505B).
Data analysis
The Agilent Feature Extraction software (version 10.7.3.1) was used to analyze the acquired array images. The quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies). After the quantile normalization of the raw data, the lncRNAs and mRNAs with flags in Present or Marginal (“All Targets Value”) in at least 2 of the 6 samples were chosen for further data analysis. The differentially expressed lncRNAs and mRNAs that exhibited statistically significant differences were identified through Volcano Plot filtering, and the differentially expressed lncRNAs and mRNAs were identified through fold-change filtering. Hierarchical clustering was performed using the Agilent GeneSpring GX software (version 11.5.1).
Results
Integrity of the sample RNA
The integrity of RNA can be assessed by electrophoresis on a denaturing agarose gel. Intact total RNA run on a denaturing gel will exhibit sharp 28s and 18s rRNA bands (eukaryotic samples). The 28s rRNA band should be approximately twice as intense as the 18s rRNA band. This 2:1 intensity ratio indicates that the RNA is intact (Figure 1).
Figure 1.
Analysis of RNA integrity and genomic DNA contamination through denaturing agarose gel. *The 28s and 18s rRNA bands are clear and intact. However, the 28s band is twice as intense as the 18s band.
Purity and concentration of the sample RNA
The NanoDrop ND-1000 was used to accurately measure the concentrations (OD260), protein contamination (ratio of OD260 to OD280), and organic compounds contamination (ratio of OD260 to OD230). The OD260/OD280 ratios of total RNA should be higher than 1.8 (Table 1).
Table 1.
Quantification and quality assurance of RNA using the NanoDrop ND-1000
| Sample ID | OD260/280 Ratio* | OD260/230 Ratio* | Conc. (ng/uL) | Volume (uL) | Quantity (ng) | QC result (Pass/Fail) |
|---|---|---|---|---|---|---|
| B93C | 1.88 | 2.04 | 352.40 | 10 | 3524.00 | Pass |
| B95C | 1.88 | 2.03 | 254.01 | 10 | 2540.10 | Pass |
| B98C | 1.90 | 2.08 | 620.42 | 10 | 6204.20 | Pass |
| B93T | 2.02 | 2.03 | 1257.93 | 10 | 75475.80 | Pass |
| B95T | 1.84 | 1.97 | 127.72 | 10 | 1277.20 | Pass |
| B98T | 1.93 | 1.98 | 521.22 | 10 | 20848.80 | Pass |
The OD260/280 ratio measured with a spectrophotometer should be closer to 2.0 for pure RNA (ratios between 1.8 and 2.1 are acceptable).
The OD260/230 ratio should be higher than 1.8.
Quality assessment of LncRNA data after filtering
Box plot
Box plots are a convenient way to quickly visualize the distributions of a dataset. These are commonly used to compare the distributions of the intensities from all samples. After normalization, the distributions of the log2-ratios among all of the tested samples were nearly the same (Figure 2).
Figure 2.
Box plot of all of the tested samples in the dataset after normalization. All six samples in the dataset were normalized, and the medians are all close to six. It is clear that the distributions of the log2-ratios are nearly the same.
Scatter plot
The scatter plot is a visualization method used to assess the LncRNA expression variation (or reproducibility) between two samples or two groups of samples. The values of the X and Y axes in the scatter plot are the normalized (log2-scaled) signal values of the samples or the average normalized (log2-scaled) signal values of a group of samples. The green lines are the fold-change lines (the default fold-change value is 2.0). The LncRNAs above the top green line and below the bottom green line are those that exhibit at least a 2.0-fold difference between the two compared samples or the two compared groups of samples. Four scatter plots are provided in the data report (Figure 3).
Figure 3.
Scatter plots for B93T vs. B93C, B95T vs. B95C, B98T vs. B98C, and T vs. C. The green lines are the fold-change lines (the default fold-change value is 2.0). The LncRNAs above the top green line and below the bottom green line are those that exhibit at least a 2.0-fold difference between the two compared samples.
Heat map and hierarchical clustering
Hierarchical clustering is one of the most widely used clustering methods to analyze LncRNA expression data. Cluster analyses arrange samples into groups based on their expression levels, which allows us to hypothesize the relationships among the samples. A dendrogram shows the relationships between the LncRNA expression patterns of different samples. Four clusters are provided in the data report (Figure 4).
Figure 4.
Hierarchical clustering of the differentially expressed LncRNAs in B93T vs. B93C, B95T vs. B95C, B98T vs. B98C, and T vs. C. The red color indicates a high relative expression, and the blue color indicates a low relative expression.
Screening of differentially expressed LncRNAs
To identify the lncRNAs that are significantly differentially expressed (Fold Change ≥ 2.0, P-value ≤ 0.05), we performed a volcano plot filtering between the two groups. To identify the differentially expressed LncRNAs, we performed a fold-change filtering between two samples (Fold Change ≥ 2.0). The expression profiles of lncRNAs were significantly different between normal bladder tissue and urothelial carcinoma of the bladder. Compared with normal bladder tissue, 1,122 lncRNAs exhibited at least a 2-fold significant difference (P < 0.05) and were thus regarded as differentially expressed lncRNAs. Of these lncRNAs, 734 increased by more than twofold, and 388 decreased by more than twofold. We also determined the lncRNAs that were upregulated or downregulated more than 10 fold in urothelial carcinoma of the bladder (Tables 2 and 3) and determined the chromosomal distribution of the differentially expressed lncRNAs (Figure 5).
Table 2.
LncRNAs that were upregulated by at least 10-fold in urothelial carcinoma of the bladder
| Sequence Name | Gene Name | Chrom. | FC Absolute | Probe Name |
|---|---|---|---|---|
| uc003 cmy.1 | AK124776 | chr3 | 26.29291 | ASHG19A3A022520 |
| C20652 | lincRNA-RAB12-1 | chr18 | 18.844774 | ASHG19A3 L0003342 |
| AK000839 | / | chr17 | 16.210579 | ASHG19A3A008508 |
| ENST00000473150 | KRT8P25 | chr3 | 15.875776 | ASHG19A3A022860 |
| ENST00000512863 | RP11-474J18.4 | chr5 | 15.131286 | ASHG19A3A027163 |
| ENST00000431957 | AC000110.1 | chr7 | 15.049078 | ASHG19A3A035014 |
| ENST00000463294 | KRT8P13 | chr3 | 14.981011 | ASHG19A3A023526 |
| ENST00000450021 | KRT8P10 | chr2 | 14.825374 | ASHG19A3A014732 |
| uc001rjo.1 | BC072678 | chr12 | 14.747757 | ASHG19A3A048481 |
| ENST00000448540 | RP11-111E2.1 | chr9 | 14.659423 | ASHG19A3A038816 |
| ENST00000504509 | RP11-789C1.1 | chr4 | 14.507504 | ASHG19A3A026300 |
| ENST00000414328 | RP11-110J1.3 | chr1 | 14.4582815 | ASHG19A3A032876 |
| ENST00000399149 | SFRS9P1 | chr21 | 14.17717 | ASHG19A3A018851 |
| ENST00000449217 | RP3-452 M16.1 | chrX | 14.110057 | ASHG19A3A040138 |
| ENST00000413425 | KRT8P18 | chr3 | 13.99092 | ASHG19A3A020870 |
| ENST00000440275 | KRT8P7 | chr11 | 13.342168 | ASHG19A3A045784 |
| ENST00000451609 | RP11-543F8.1 | chr10 | 13.288165 | ASHG19A3A042424 |
| EC495588 | lincRNA-RCN2 | chr15 | 12.822171 | ASHG19A3 L0002752 |
| ENST00000367074 | GS1-309P15.4 | chrX | 12.674266 | ASHG19A3A040786 |
| ENST00000424882 | GS1-309P15.3 | chrX | 12.63172 | ASHG19A3A039699 |
| ENST00000485873 | RP4-621B10.3 | chr1 | 12.627898 | ASHG19A3A048157 |
| ENST00000398745 | KRT8P26 | chr11 | 12.41178 | ASHG19A3A046607 |
| ENST00000511504 | RP11-790I12.1 | chr4 | 12.265042 | ASHG19A3A025622 |
| nc-HOXB4-176+ | nc-HOXB4-176 | chr17 | 12.118851 | CUST_54_PI426075208 |
| ENST00000454391 | AL358913.1 | chr14 | 11.904219 | ASHG19A3A050730 |
| ENST00000497538 | RP11-111F10.2 | chr3 | 11.498124 | ASHG19A3A023430 |
| ENST00000392131 | AC079305.6 | chr2 | 11.317965 | ASHG19A3A016645 |
| ENST00000510090 | RP11-618I10.2 | chr4 | 11.099051 | ASHG19A3A025620 |
| ENST00000434750 | AC108171.5 | chrX | 10.894489 | ASHG19A3A041704 |
| ENST00000453086 | RP5-1091N2.3 | chrX | 10.1777935 | ASHG19A3A040026 |
Table 3.
LncRNAs that were downregulated by at 10-fold in urothelial carcinoma of the bladder
| Sequence Name | Gene Name | Chrom. | FCAbsolute | Probe Name |
|---|---|---|---|---|
| nc-HOXB9-206- | nc-HOXB9-206 | chr17 | 272.16144 | ASHG19A3H0000077 |
| ENST00000426074 | RP11-160A10.2 | chr6 | 52.299904 | ASHG19A3A032028 |
| nc-HOXA11-86+ | nc-HOXA11-86 | chr7 | 37.83364 | CUST_4_PI426075208 |
| AL109700 | / | chr15 | 36.111774 | ASHG19A3A052515 |
| AK123324 | / | chr8 | 28.818373 | ASHG19A3A035994 |
| nc-HOXD10-7- | nc-HOXD10-7 | chr2 | 28.742731 | ASHG19A3H0000169 |
| BC042023 | / | chr1 | 27.417946 | ASHG19A3A047528 |
| AK026384 | / | chr2 | 26.26481 | ASHG19A3A016486 |
| nc-HOXB9-205- | nc-HOXB9-205 | chr17 | 25.701212 | ASHG19A3H0000076 |
| ENST00000421606 | CES4 | chr16 | 24.578844 | ASHG19A3A054608 |
| nc-HOXD12-3- | nc-HOXD12-3 | chr2 | 21.637152 | ASHG19A3H0000174 |
| uc002tjx.3 | AY343891 | chr2 | 20.35203 | ASHG19A3A014229 |
| ENST00000416700 | RP11-58A12.3 | chr9 | 17.737352 | ASHG19A3A038722 |
| AL109696 | / | chr15 | 15.587008 | ASHG19A3A052517 |
| uc010 mam.2 | AF131817 | chr8 | 15.372687 | ASHG19A3A035947 |
| uc003tcg.2 | AK124304 | chr7 | 15.336274 | ASHG19A3A034154 |
| ENST00000442797 | AC007064.21 | chr22 | 15.284027 | ASHG19A3A020031 |
| AL390167 | / | chr13 | 14.648443 | ASHG19A3A049319 |
| ENST00000514910 | RP11-451F20.1 | chr4 | 14.038315 | ASHG19A3A026355 |
| uc003xjb.2 | BC034319 | chr8 | 13.934998 | ASHG19A3A036526 |
| CF129145 | lincRNA-LOC100506581-1 | chr16 | 13.665986 | ASHG19A3 L0000894 |
| uc001awc.1 | AK055853 | chr1 | 13.640614 | ASHG19A3A012055 |
| AK125472 | / | chr13 | 13.590306 | ASHG19A3A049853 |
| uc001 gla.1 | CR621436 | chr1 | 12.739795 | ASHG19A3A035511 |
| uc004ags.1 | AK130904 | chr9 | 12.102253 | ASHG19A3A038744 |
| AF087976 | / | chr3 | 11.665831 | ASHG19A3A022767 |
| G65639 | chr5 | 10.440295 | ASHG19A3A026776 | |
| AK307235 | lincRNA-SRD5A2 | chr2 | 10.403127 | ASHG19A3 L0001181 |
| ENST00000439715 | RP11-282E4.1 | chr9 | 10.397835 | ASHG19A3A038699 |
| ENST00000434499 | RP11-145A3.4 | chr1 | 10.215157 | ASHG19A3A040793 |
Figure 5.
Chromosomal distribution of differentially expressed lncRNAs. Most of the differentially expressed lncRNAs are distributed on chromosomes 1, 2, 3, 4, 6, and X.
Discussion
The large amount of clinical observations and experimental studies on lncRNAs demonstrates that lncRNAs play an important role in the tumorigenesis and progression of urologic oncology, including bladder cancer. It has been reported that there is a significant difference in the expression of lncRNAs between normal tissue and tumor tissue [22] and between different stages of the same tumor [23]. Although its function has not been deeply studied, the mechanism through which lncRNAs participate in or influence the tumorigenesis and progression remains unclear, but its potential value in future studies is self-evident. We have found that the majority of lncRNAs are highly conserved and have specific gene sequences. These are widely expressed in both normal cells and tumor cells of human being, have many copies, and are distributed in specific chromosomal loci and tumor-associated regions. Certain lncRNAs are closely associated with the tumorigenesis and progression of bladder tumors and thus may contribute to the prevention, diagnosis, and treatment of bladder tumors in the future.
Research on chromosomal distribution of lncRNAs in bladder cancer
It is well recognized that cancer is a hierarchical disease that is caused by a synergy of oncogenes, tumor-suppressor genes, changes in repair genes, and other factors. Recent studies have found that tumorigenesis is not only related to the activation of oncogenes and the inactivation of tumor-suppressor genes but also connected with gene methylation, microsatellite genetic abnormalities, and telomere abnormal expression, which can occur on any chromosome. According to the abovementioned data, we hypothesize that lncRNAs may also participate in these changes. Our studies have shown that the differentially expressed lncRNAs in bladder urothelial carcinoma are distributed on each of the chromosomes, although the majority of lncRNA are found on chromosomes 1, 2, 3, 4, 6, and X. In the tumorigenesis and progression of bladder cancer, the chromosomes show chromosomal abnormalities, which are not confined within a particular chromosome. All of the chromosomes, including the X and Y chromosomes, can experience different quantities and degrees of changes in bladder cancer, and our experimental results are consistent with this assertion. The chromosomal abnormalities include gene amplifications, gene rearrangements, gene deletions, and numerical and structural anomalies. It has been reported in the literature that chromosomal abnormalities are mainly distributed in chromosomes 3, 7, 8, 9, 11, 13, 16, 17, and Y in bladder cancer [24-26], and we hypothesize that the degrees and quantities of these abnormalities are associated with the grade and stage of the tumor, the depth of invasion, and the recurrence of tumors. We also took the experimental methods into account because the main experimental method the is currently used to detect chromosomal abnormalities is FISH technology, which emerged in the late 1980s and is a non-radioactive in situ hybridization method that was developed based on radioactive in situ hybridization. The basic principle of FISH is the use of a fluorescent dye-labeled specific nucleic acid as a probe, which, based on base complementary, will specifically bind a nucleic acid in the sample to form a hybrid double-stranded nucleic acid. Fluorescence microscopy is then used to identify and count the fluorescent signals, which allows the qualitative positioning and quantitative analysis of the chromosomes in the sample. In addition to the differences in the experimental methods used, we took into consideration the possible relationship with the sample size. The objective of our experiments was to screen the differentially expressed lncRNAs in bladder cancer to identify genes that are specific to bladder cancer. We aim to screen large samples though microarray technology, but were limited by the high cost of microarray technology. Hence, the methodology, the sample size, and the intrinsic factors of the tumors themselves, including the different stages and grades, are likely to cause differences in the distribution of chromosomal aberrations. However, it is clear that the tumorigenesis and the progression of bladder cancer is not the result of a single chromosomal aberration but of multiple chromosomal abnormalities.
Research on differentially expressed lncRNAs in bladder cancer
In the current literature on bladder cancer, lncRNAs are rarely mentioned. The only relevant lncRNA that has been reported is urothelial cancer-related genes (UCA1), and this lncRNA was first reported in 2006 by Wang [27]. This lncRNA was initially considered an urothelial cancer-specific gene, but it was later also found in placental, gallbladder, and epithelial tissue [28-30]. UCA1 is positioned within the cytoplasm and is located in human chromosome 19p13.12, which has three exons and two introns. There are three different splice bodies with lengths of 1.4 kb, 2.2 kb, and 2.7 kb [31], and the first splice body was found mainly in bladder urothelial carcinoma. Wang [32] first used suppression subtractive hybridization to analyze BLS-211 and BLZ-211 cells and obtained a 1,442-bp UCA1 gene. The function of this lncRNA may be associated with the regulation of the downstream molecular processes of protein synthesis. The UCA1 gene can increase the malignant phenotype of the bladder urothelial carcinoma cell line BLS-211 and increases its proliferation in vitro. In addition, this gene significantly increased the invasiveness and drug resistance markedly this cell line, causes significant tumorigenesis in nude mice, upregulates the gene expression of WNT6, CYP1A1, and AURPK, and downregulates the expression of MBD3 and SRPK1 [33]. In the detection of urinary sediments of different pathological grades in urothelial carcinoma patients, it was found that this gene expression pattern is closely related to G2-G3 superficial bladder cancer: 40% similar to G1, 90.9% similar to superficial G2, 64.3% similar to invasive G2, 91.7% similar to superficial G3, and 100% similar to invasive G3 [27]. In our experiment, UCA1 was expressed, but the data analyses demonstrated that the two groups did not exhibit differential expression of UCA1, likely due to the sample size, pathological differences, methodology used, and other factors. Microarray technology is highly sensitive; however, whether a highly expressed lncRNA or a related RNA is our bladder cancer-specific target remains controversial. Further validation, including RT-PCR analyses, is required, and a variety of tissues (not just bladder cancer tissue) need to be verified to ensure that the target is specific to bladder cancer.
At present, research on lncRNAs is just beginning, and many of the features of lncRNAs, including their mechanism of action and their relationship with tumor development, are still unclear. Our experiment obtained differentially expressed lncRNAs that have not been previously reported in the literature. These include some lncRNAs that were upregulated, such as AK124776, lincRNA-RAB12-1, KRT8P25, RP11-474J18.4, AC000110.1, KRT8P13, KRT8P10, and BC072678, and some lncRNAs that were downregulated, such as nc-HOXB9-206, RP11-160A10.2, nc-HOXA11-86, nc-HOXD10-7, nc-HOXB9-205, CES4, and nc-HOXD12-3. In future research, we will continue to search for bladder cancer-specific or -related lncRNAs. This study sets the basis for future research on lncRNAs in bladder carcinoma.
Acknowledgements
This work was supported by the Shanghai Education Commission Research and Innovation projects (No. 12ZZ034).
Disclosure of conflict of interest
None.
Abbreviations
- UCC
urothelial cell carcinoma
- LncRNA
long noncoding RNA
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