Background
Bladder cancer (BC) is the most common malignancy of the urinary tract worldwide [
1]. About 75% of patients are classified as non-muscle-invasive BC (NMIBC), while the rest are muscle-invasive BC (MIBC) [
2]. NMIBC features a high recurrence rate and MIBC patients usually have a poor prognosis because of recurrence or metastasis [
2,
3]. Cystectomy is the main treatment of MIBC and has a 5–15% probability of pelvic recurrence that most often occurs within 6–18 months after surgery. Pelvic recurrences usually carry a poor prognosis, despite treatment, with median survival from 4 to 8 months [
4]. The poor prognosis of BC is partially due to lack of an effective non-invasive means for early screening. At present, cystoscopy and pathological biopsy are the golden standard for diagnosing BC [
5]. However, the invasiveness of cystoscopy limits its application as a regular screening tool for BC. Currently urine cytology is the most commonly used non-invasive diagnosis tool for BC, but is limited by low sensitivity [
6]. Therefore, exploration of effective non-invasive biomarkers for screening BC can play a pivotal role in improving the prognosis and quality of lives of BC patients.
MicroRNAs (miRNAs) are endogenous 21-23 nt small non-coding RNAs with a length of approximately 21–23 nucleotides, which are capable of suppressing gene expression through post-transcriptional regulation. miRNAs are involved in various physiological and pathological procedures [
7], which includes but not limit to, embryo development [
8], oncogenesis [
9,
10], and immune regulation [
11]. miRNAs have been isolated and identified in many kinds of biofluids, such as plasma, serum, and urine [
12], suggesting the potential of miRNAs as minimally invasive biomarkers of cancer [
13]. But the instability of free miRNAs in biofluids hinders its application in clinical practice.
Exosomes are described as small membrane vesicles with a diameter of approximately 30–150 nm derived from cell endosomes [
14]. They can be secreted into nearly all body fluids, including blood and urine, by nearly all kinds of cells [
15]. The membrane construction contained a selection of miRNAs, mRNAs, lncRNAs, proteins, and lipids [
16,
17]. Exosomes can act as messengers in cell-to-cell communication by transferring contained molecules and play important roles in tumorigenesis, progression and metastasis [
18].
There is increasing evidence suggested the potential role for exosomal miRNA in early diagnosis of many diseases [
19,
20]. However, systematic screening of urinary exosomal miRNA serving as BC biomarkers has not yet been studied. The purpose of this study is to identify urinary exosomes derived miRNA biomarkers as a non-invasive method to discriminate BC and clarify the mechanism of miR-93-5p in BC cells.
Materials and methods
Patients enrollment and sample collection
A total of 12 patients with BC (six NMIBC and six MIBC) and 4 non-cancerous participants (as healthy control) were recruited from Nanfang Hospital of Southern Medical University (Guangzhou, China) between March 2018 and December 2019 as the discovery set for discovering miRNAs suitable for biomarkers. An additional 104 participants (53 BC and 51 healthy control) were enrolled for validation. Written informed consent was obtained from each participant. This study was approved by the ethics committee of Nanfang Hospital. Midstream urine samples from each BC patients and healthy controls with 50 mL were collected, complying with the following criteria: urine samples from BC patients were collected before any antitumor therapies, such as surgery, chemotherapy or radiotherapy; urine samples of healthy controls were acquired from people who went through a medical check-up and showed no disease; and all these participants, showed no evidence of disease in other organs. BC was diagnosed based on histopathological findings. The tumor stage and grade were decided complied with the tumor-node metastasis (TNM) staging system and the WHO 2004 grading scheme, respectively [
21]. Urine samples were immediately centrifuged at 3000 g for 20 min at 4 °C to remove cell debris, and the supernatant fluids were then collected and stored at − 80 °C until exosome extraction.
Urinary exosome isolation
We conducted differential ultracentrifugation to isolate exosomes according to the standard method with a slight modification as previously depicted [
22]. The aforementioned cell-free supernatant was centrifuged at 17,000 g for 30 min. Subsequently, supernatant was obtained and filtered with 0.22-μm filters (Millipore, Burlington, MA, USA) to remove microvesicles (200–1000 nm in diameter) and apoptotic bodies (800–5000 nm in diameter). Eventually, the supernatant that originated from the previous procedure was centrifuged at 110,000 g for 70 min at 4 °C in an ultracentrifuge (Beckman Coulter, Miami, FL, USA) to pellet exosomes from urine. After the supernatant was extracted, the exosomal pellet was suspended with 200 μL of phosphate-buffered saline (PBS) again.
Transmission electron microscopy (TEM)
We used TEM to observed and identified the structure of isolated exosomes as previously reported and manufacturer’s protocols [
23]. A total of 20 μL exosomes-enriched solution was placed on a copper mesh and incubated at room temperature for 10 min. After washing with sterile distilled water, the exosomes was contrasted by uranyloxalate solution for 1 min. The sample was then dried for 2 min under incandescent light. The copper mesh was observed and photographed under a JEM 1400 transmission electron microscope (JEOL, Tokyo, Japan).
Nanoparticle tracking analysis (NTA)
Exosomes isolated from urine were processed for nanoparticle tracking analysis (NTA) with NanoSight NS300 instrument (Malvern, UK). Briefly, exosomes were diluted in 1 mL PBS and mixed well, and then the diluted exosomes were injected into the NanoSight NS300 instrument (Malvern, UK). Instrument settings were set according to the manufacturer’s software manual. Particles were automatically tracked and sized based on the in-build NanoSight Software NTA 3.1 Build 3.1.46. Filtered PBS was used as a control.
Western blot analysis
Total protein was extracted in RIPA lysis buffer (89,900, Thermo Fisher Scientific, Waltham, MA, USA). Equal loading of extracted protein was denatured in 5× sodium dodecyl sulfonate (SDS) buffer and subjected to western blot analysis (10% SDS-polyacrylamide gel electrophoresis; 50 μg protein/lane) using rabbit polyclonal antibody CD63 (abs-134,386, Absin, Shanghai, China), TSG101(abs-122,785, Absin, Shanghai, China), BTG2(ab-24,460, Abcam, UK), Calnexin (ab-133,615, Abcam, UK) and GAPDH (abs-133,958, Absin, Shanghai, China). The proteins of interest were detected on a gel imaging system using ECL western blotting substrate (Thermo Fisher Scientific, Waltham, MA, USA) and band density was analysed with ImageJ software.
RNA extraction and qRT-PCR
Total RNA extraction from exosomes, tissues and cultured cells was performed with the Trizol Reagent (15,596,026, Invitrogen, Carlsbad, CA, USA) as previously reported [
24]. The resulting RNA pellet was stored at − 80 °C until further analysis. RNA was quantified and assessed by NanoDrop® ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA). The expression of three mature miRNAs namely hsa-miR-93-5p, hsa-miR-516a-5p, and hsa-miR-940 (Table I) was quantified using TaqMan single® microRNA assays (442,975, Applied Biosystems®) in accordance with the manufacturer’s protocol. The same amount of
Caenorhabditis elegans cel-39 miRNA was spiked into each exosomes sample as an external calibration for RNA extraction, reverse transcription, and miRNA amplification. Real-time PCR was performed with ABI 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative gene expression was calculated using the 2
-□□Ct method [
25] and normalized to spike-in control cel-miR-39 or endogenous control U6 snRNA.
Library preparation and sequencing
High throughput sequencing technology for exosomal miRNAs from urine was performed as manufacturer’s recommendations and previously reported [
23,
26]. For small RNA libraries, a total amount of 2.5 ng RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEB Next Multiplex Small RNA Library Prep Set for Illumina (NEB, Ipswich, MA, USA) following the manufacturer’s recommendations. Index codes were added to attribute sequences to each sample. Adapter Ligated RNA was mixed with ProtoScript II Reverse Transcriptase, Murine RNase Inhibitor, First Strand Synthesis Reaction Buffer (NEB, Ipswich, MA, USA) and incubated for 60 min at 50 °C. We mixed the purified PCR product (25 μL) with 5 μL of Gel Loading Dye, loaded 5 μL of Quick-Load pBR322 DNA-MspI Digest in on the 6% PAGE 10-well gel, run the gel for 1 h at 120 V. For miRNA, the bands corresponding to ~ 150 bp were isolated. At last, the library quality was assessed with both the 2100 Bioanalyzer System (Agilent, Santa Clara, CA, USA) and qPCR. After cluster generation, the libraries were sequenced on an Illumina Hiseq X ten platform and 150 bp paired-end reads were generated.
Differential expression analysis of miRNA
Differential expression analysis of exosomal miRNAs was performed as previously reported [
23]. With the help of software Bowtie, clean reads were aligned and compared with sequences in databases including Silva, GtRNAdb, Rfam, and Repbase respectively. Reads with more than 10%N, low quality, length > 32 nt/< 16 nt, or trimming 3′ adapter from the end of reads (no mismatch) were filtered. After filtering unwanted sequences, such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA), remaining reads were compared with miRNAs from miRbase and Human Genome (GRCh38) to identify known miRNAs as well as the prediction of new miRNAs. Reads counts were generated according to the mapping results of miRDeep2, which was used to calculate TPM. Level 3-normalized miRNA expression data for 410 bladder cancer patients were obtained from TCGA (
https://portal.gdc.cancer.gov/) by using R language package TCGAbiolinks. Datasets from 19 solid normal tissue samples of bladder urothelial as non-cancerous control set were also obtained. Then we used the TMM method [
27] in edgeR to normalize the TPM of miRNA. A miRNA was regarded as differentially expressed if it exhibited |log2(Fold Change)| > 1(
p < 0.05).
Target gene prediction, GO/KEGG pathway enrichment analysis and PPI network
Potential target genes of selected miRNA were predicted by miRWALK2.0, an online archive of data on miRNA-target interactions [
28] for further analysis. In total, 12 servers with miRWalk, miRMap, MicroT4, miRNAMap, TargetScan, PICTAR2, miRBridge, PITA, miRanda, RNAhybrid, miRDB, RNA22 were used. Only those genes projected by more than six of the servers were recognized as target genes. Since miRNAs could down-regulated the expression of target genes, the low-expressed genes in bladder cancer were acquired through bioinformatic analysis from public data from TCGA database. The overlapping genes among the down-regulated genes in BC and the predicted target genes, were viewed as promising targets of selected miRNA in BC. The Gene Ontology (GO) analysis, which include biological processes (BPs), cellular components (CCs), and molecular functions (MFs), were conducted by clusterProfiler R package. The functional annotation of the underlying target genes was then elucidated by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with clusterProfiler R package. In addition, a Protein-Protein Interaction (PPI) network was constructed to reveal the hub genes of the potential target genes on STRING, a web portal for undermining the integrated function of multiple genes [
29].
Cell lines and cell culture
The human BC cell lines T24, UM-UC-3, as well as one normal bladder cell line SV-HUC-1, were purchased from the Shanghai Institute of Cell Biology, Shanghai, China. These cell lines were cultured according to manufacturer’s recommendations and previously reported. Cell lines were maintained in Roswell Park Memorial Institute 1640 medium (RPMI1640; Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Biological Industries, Cromwell, CT, USA), under a humidified atmosphere of 5% CO2 at 37 °C. The cell culture medium was changed every 2–3 days, and the cells were passaged with 0.25% trypsin-EDTA (Gibco, Carlsbad, CA, USA) and grown to 90% confluence.
Reagents and transfection
The hsa-miR-93-5p mimic (miR-93-5p mimic; miRBase accession MIMAT0000093; sense: 5′-CAAAGUGCUGUUCGUGCAGGUAG-3′), the negative control (NC) of the mimic duplex (NC, sense:5′-UUCUCCGAACGUGUCACGUTT-3′), the hsa-miR-93-5p inhibitor (miR-93-5p inhibitor; sense: 5′-CACUUAUCAGGUUGUAUUAUAA-3′) and the negative control duplex of the inhibitor (inhibitor NC, sense:5′-CAGUACUUUUGUGUAGUACAA-3′) with no significant homology to any known human sequences were used for gain-of-function studies. The RNA duplexes were chemically synthesized by GenePharma, Shanghai, China. Oligonucleotide transfection was performed using Lipofectamine 2000 reagents (11,668,019, Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol.
Dual-luciferase reporter assay
Dual-luciferase reporter assay was performed as previously reported [
30]. Olgonucletide pairs that contained the desired miR-93-5p target region or mutant target region were designed and ordered from GenePharma, Shanghai, China. After annealing, these double-stranded segements were inserted into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA), between the SacI and SalI sites. The insertions were verified by sequencing. Dual-luciferase assays were performed using 1 × 10
4 T24 cells per well in a 96-well plate (Corning, Acton, MA, USA). After the cells attached for 8 h, they were cotransfected with 50 ng of miRNA mimics or control miRNA. After 48 h, a Reporter Assay System Kit Pierce™ (16,186, Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the luciferase activity. There were three replicates for each transfectant. Firely luciferase activity was normalized to constitutive Renilla luciferase activity.
Cell growth/cell viability assay (cell count Kit-8 assay)
Cell Count Kit-8 assay was performed to evaluate the cell viability as previously reported [
30,
31].T24 or UM-UC-3 cells were plated in 96-well plates with ~ 4 × 103 cells per well. After overnight incubation, the cells were transfected with the RNA duplex (miR-93-5p mimic,miR-93-5p inhibitor, or NC) for 2–3 days with concentrations 50 nM. At different time points, the medium was removed and WST-8 (Dojindo Laboratories, Kumamoto, Japan) was added to each well. After the 96-well plate was incubated at 37 °C for 1 h, the absorbance of the solution was measured spectrophotometrically at 450 nm with an MRX II absorbance reader (Dynex Technologies, Chantilly, VA, USA).
Cell migration and invasion assay
The cell migration and invasion assay were performed according to the standard method with a slight modifications as previously reported [
30,
31]. For the invasion assay, the inserts were coated with Matrigel (BD Bioscience, Franklin Lakes, NJ, USA) on the upper surface. After transfection, 8 × 104 cells were suspended in 0.2 ml serum-free medium and added to the inserts. Then, 0.6 ml RPMI-1640 medium with 10% FBS was added to the lower compartment as a chemoattractant. After incubation at 37 °C for 24 h, the cells on the upper surface of the membrane were carefully removed using a cotton swab and cells on the lower surface were fixed with 100% methanol and stained with 0.1% crystal violet. Five visual fields of 200× magnification of each insert were randomly selected and counted under a light microscope (Olympus, Japan).
Statistical analysis
Statistical tests were performed using R 3.5.1 (
www.r-project.org). Data are presented as median (interquartile interval). All tests were two-tailed and False Discovery Rate (FDR) was controlled for multiple comparisons. The differences in the expressions of UE-derived miRNAs between BC patients and healthy controls were assessed by non-parametric Mann–Whitney U test. Diagnostic accuracy of candidate miRNAs or their combinations was assessed by receiver operating characteristic (ROC) curves analysis. Correlation analysis was conducted by Pearson’s correlation method. Survival curves were generated by Kaplan–Meier method, and the difference was compared by log-rank test. Packages plyr and reshape2 were used for data sorting and restructuring. VennDiagram, pheatmap, and ggplot2 were used for visualization of results. A
P value < 0.05 was considered as statistically significant.
Discussion
Compared with other clinical samples from patients such as blood, urine has its unique advantages in clinical application: acquired non-invasively and easily accessible, which makes it a suitable source of biomarkers for many diseases [
37]. Exosomes, extracellular microvesicles with diameter 30-150 nm, secreted by nearly all kinds of cells including cancer cells exist in biofluids, has been identified to contain bioactive molecule such as RNA (mRNA, miRNA, lncRNA), protein and lipid from original cells [
38]. Moreover, the membrane structure of exosomes protect the contained molecule from degradation by enzymes [
22]. Several studies have indicated that compared with cell-free urine, miRNAs were much more enriched and stable in urine-derived exosomes, and aberrant expression of particular miRNAs in exosomes might reflect the change in biological processes of disease [
39]. Thus, urine-derived exosomal miRNA has attracted much attention from researchers as a new diagnostic tool to screen for disease [
20,
40]. For instance, researchers had found that the urinary exosomes-derived miR-181a in patients with chronic kidney disease was significantly decreased compared with health control, making it a potential indicator for CKD diagnosis. As for bladder cancer, urine-derived exosomes had also been investigated as biomarkers. Zhan [
41] and et.al developed a urinary exosome-derived lncRNA panel (MALAT1, PCAT-1 and SPRY4-IT1) for diagnosis and recurrence prediction of bladder cancer in a cohort consist of 368 urine samples. However, the sample the signature of exosomal miRNAs and its performance in diagnosis in urine for BC patients has not been sufficiently examined.
In the present study, a comprehensive analysis of urinary exosome-derived miRNA profile of BC patients and non-cancerous controls was performed with high throughput sequencing combined with RT-qPCR assays. We discovered that the expression profile of miRNAs in urinary exosomes from BC patients was markedly different from that of healthy controls, and identified 51 miRNAs up-regulated and 22 miRNAs down-regulated in BC samples compared to controls. In order to narrow the range of candidate miRNAs into miRNAs associated with development of bladder cancer to maximize the success rate of urinary exosomes biomarker verification, we identified the total 61 differentially expressed miRNAs (40 up-regulated and 21down-regulated) between MIBC and NMIBC. Then we selected miRNAs (106 up-regulated, 26 were down-regulated) that were also differentially expressed in the TCGA database as biomarker candidates. The reason why we compare our data side by side with differentially expressed miRNAs identified from TCGA is based on the assumption that miRNAs differentially expressed in bladder tumor is more likely to be a valid exosome-associated biomarker of BC. Finally, after the intersection, miR-93-5p, miR-516a-5p and miR-940(all up-regulated) were selected for further validation. By RT-qPCR assay validation arranged in an independent cohort, miR-93-5p and miR-516a-5p were validated to be significantly and steadily increased in BC patients. The ROC analysis showed that miR-93-5p and miR-516a-5p had relatively promising AUCs for BC diagnosis. In the BC cohort, RT-qPCR assay showed that miR-93-5p was significantly elevated in MIBC patients compared with NMIBC and ROC result suggested miR-93-5p exhibited a promising AUC for distinguishing MIBC and NMIBC. Moreover, correlation analysis also showed that urinary exosomal miR-93-5p expression level was significantly related with TNM stage (Ta-T1 vs T2-T4) of BC patients. These results suggested that urinary exosomal miR-93-5p might have the ability to distinguish MIBC and NMIBC. It must be noted that we did not observe a significant difference in the expression level of candidate miRNAs among BC patients with different pathological types. Considering that the sample size was small in our study, more specimens will be needed to clarify whether these urinary exosomal miRNAs have the ability to distinguish different pathological types.
The studies of miR-516a-5p were rare and understanding of miR-516a-5p remained uncertain. In this study, we discovered that miR-516a-5p was overexpressed in bladder cancer tissues and urinary exosomes from BC patients. The urinary exosome-derived miR-516a-5p had the potential to be a non-invasive biomarker for BC. Ye [
42] and his colleagues had found miR-516a-5p might act as a tumor suppressor and inhibit the proliferation of non-small cell lung cancer by targeting HIST3H2A gene. More experiments are needed to explore the mechanism of miR-516a-5p in BC.
MiR-93-5p has been found enriched in various types of tumors [
43,
44] and mainly played an oncogenic role in the development of malignant neoplasm, including bladder cancer [
45]. Sun [
43] and et.al had found that miR-93-5p was up-regulated in cervical cancer cells and promotes cancer progression by down-regulating THBS2/MMPS signal pathway. In bladder cancer, Jiang [
45] and et.al had indicated that miR-93 plays an oncogenic role by inhibiting expression of PEDF to promote cancer cells proliferation and invasion through, which was partially in accordance with our bioinformatic analysis results. In our bioinformatic analysis, we firstly predicted and selected the candidate target genes of miR-93-5p via mirWalk and TCGA database. GO enrichment and KEGG pathway analysis were performed to explore the possible mechanisms under miR-93-5p and its target genes in bladder cancer. According to the GO/KEGG analysis and a series studies reported before, we assumed that miR-93-5p might act as a tumor promoter in bladder cancer and its target genes were mainly enriched in proteoglycans in cancer, EGFR tyrosine kinase inhibitor resistance, FoxO signaling pathway, PI3K-Akt signaling pathway and MAPK signaling pathway. These pathway might play an important role in the mechanisms under miR-93-5p in development of bladder cancer. PPI network and survival analysis showed that BTG2 was the hub target gene of miR-93-5p and associated with the prognosis of BC patients. Therefore, we conducted a series of in vitro experiment to verified the bioinformatic analysis results. In this study, RT-qPCR validation assay suggested that the expression of miR-93-5p was both significantly up-regulated in BC tissues and human BC cells compared to that in adjacent non-cancerous tissues and normal cells. The CCK-8 and transwell assay showed that overexpression of miR-93-5p promoted the proliferation, migration and invasion of bladder cancer cells. At the same time, we demonstrated that there was an inverse correlation between miR-93-5p and BTG2 expression. Our findings were consistent with those from previous studies and our bioinformatic analysis.
B-cell translocation gene2(BTG2), the first identified gene in BTG/TOB gene family [
46], is engaged in many biological processes in tumor development course. The BTG2 is located on band 2, region 3 of the long arm of chromosome 1 and its mRNA translates 158 amino acids. Biological functions of BTG2 mainly includes the following aspects: a) DNA damage repair. Previous studies had demonstrated that DNA damage stimulates p-53 to activated BTG2 expression, which results in down-regulation of cyclinD1 and inhibition of G1/S transition via pRB pathway [
47]. b) Anti-migration. BTG2 had been reported to inhibit Src-FAK (focal adhesion kinase) signaling by downregulating reactive oxygen species (ROS) generation and therefore exert a negative effect on cancer cell metastasis [
48]. c) Anti-proliferation. Up-regulation of BTG2 activated by p53-dependent or p53-independent NF-κB signaling pathway inhibits cancer cell proliferation [
49], and BTG2 also negatively regulates jAK2/STAT3 signaling to exert an anti-proliferation effects on cells [
50]. d) Cell death regulation. Ryu et al. [
51] found that overexpression of BTG2 induces G2/M arrest and cell death by inhibiting cyclin B1-Cdc2 binding. e) Differentiation regulation. Several studies demonstrated that BTG2 gene participates in the development and differentiation of nerve cells [
52]. Farioli-Vecchioli et.al [
53] demonstrated that PC3/Tis21 knockout mice display impaired terminal differentiation of hippocampal granule neurons and defective contextual memory. In a word, BTG2 plays important roles in cell differentiation, proliferation, DNA damage repair and cell death.
BTG2 is expressed in different kinds of organs and tissue, such as spleen, lung, prostate, stomach, pancreas and so on [
54]. A series of studies demonstrated that BTG2 mainly acted as a tumor suppressor [
33,
34,
48] in cancer. Zhang [
55] et.al demonstrated that BTG2 is down-regulated in gastric cancer tissue, and overexpression of BTG2 suppresses the proliferation and migration of gastric cancer cells. Wei [
36] et.al demonstrated that overexpression of BTG2 inhibits the protein expression of cyclin D1, MMP-1, and MMP-2, and inhibit the growth, proliferation, and invasiveness of the A549 human lung cancer cell line. Several studies demonstrated relationship between BTG2 and miRNAs. Liu [
56] et.al discovered that miR-21 enhances the proliferation and suppresses the apoptosis of laryngeal cancer cells via targeting and downregulating BTG2. Xie [
7] et.al demonstrated that miR-6875-3p promotes the proliferation, invasion and metastasis of hepatocellular carcinoma by mediating BTG2/FAK/Akt pathway. In this study, we reveal that BTG2 act as s tumor suppressor in bladder cancer and overexpression of miR-93-5p inhibited BTG2 and then promote the proliferation, migration and invasion of bladder cancer cell. But it still needs more experiments to investigate the concrete signaling pathway and mechanisms between miR-93-5p and BTG2 in bladder cancer.
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