Background
Bladder cancer (BC) is one of the most common malignancies in the genitourinary system, with approximately 400,000 new cases diagnosed annually and over 165,000 deaths [
1]. Although treatment such as transurethral resection and intravesical chemotherapy may be successfully applied for non-muscle-invasive bladder cancer (NMIBC), the unfavorable prognosis and high rate of recurrence and metastasis of muscle-invasive bladder cancer (MIBC) result in a 5-year survival rate of approximately 60% [
2]. Improved understanding of the mechanisms of BC metastasis and progression will thus likely improve the effectiveness of therapy in patients with advanced stage BC.
Circular RNA (circRNA) are a class of non-coding RNA transcripts that are generated from backsplicing of precursor mRNA [
3]. circRNAs are characterized by covalently closed continuous loops without 5′ or 3′ polarities, and are more stable and more resistance to digestion with RNase R than liner transcripts [
4]. Studies have reported that circRNAs regulate various biologic processes such as gene expression, transcription, cell proliferation, and apoptosis [
5,
6]. In addition, abnormal expression of circRNAs has been found to be involved in the progression of a variety of human cancers [
7,
8]. For example, circ-Foxo3 prevents mouse double-minute 2 (MDM2) from inducing Foxo3 ubiquitination and degradation, resulting in increased levels of Foxo3 protein and tumor cell apoptosis [
9]. A recent study demonstrated that circ-TTBK2 decreases miR-217 expression and promotes glioma malignancy by regulating the miR-217/HNF1β/Derlin-1 pathway [
10]. In bladder cancer, several circRNAs have been shown to act either as a tumor suppressor or an oncogene via different targets [
11,
12].
In the present study, we identified a novel circRNA designated circ_0008532 as an oncogene in bladder cancer. Expression of circ_0008532 is significantly upregulated in bladder cancer tissues and cell lines, and is positively associated with bladder cancer progression by sponging miR-155-5p/miR-330-5p to influence the expression of MTGR1 and the activity of Notch signaling. Circ_0008532 may exert regulatory functions and serve as a target for bladder cancer treatment.
Methods
Cell culture
Primary cultures of normal bladder urothelial cells (NBUCs) were established from fresh patient specimens. The uroepithelial cell SV-HUC-1 and bladder cancer cell lines (5637, UM-UC-3, TCCSUP, T24, EJ, SCaBER, T24T, J82, SW780) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All these cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco).
Tissue specimens
Ten bladder cancer tissues and matched adjacent non-tumor bladder tissues were obtained from the Department of Urology, Huazhong University of Science and Technology affiliated Union Hospital and stored in liquid nitrogen pending use. To select adjacent non-tumor bladder tissues, grossly normal mucosa from the resection margin most distant from tumor was carefully excised and subjected to frozen section evaluation in order to exclude dysplasia and the presence of carcinoma cells. The urothelium and submucosal layers of an adjacent area was then carefully peeled off and placed immediately in liquid nitrogen.
RNA extraction and quantitative real-time PCR (real-time qPCR)
Total RNA was extracted from cells and fresh tissue using the Trizol (Invitrogen) kit according to the manufacturer’s instructions, and was reverse transcribed using the RevertAid First Strand cDNA SynthesisKit (Thermo Scientific, MA, USA). Subsequently, real-time qPCR was performed on a StepOne Plus real-time PCR system (Life Technologies, Carlsbad, CA). GAPDH was used as an internal control. The sequences of primers are provided in the Additional file
1: Table S1. The 2 − ΔΔCT method was used to calculate relative expression of mRNA.
Western blotting
Cells and tissue samples were lysed in RIPA lysis buffer, and protein concentrations were determined using a BCA Protein Assay Kit (Thermo Scientific, MA, USA). Cell/tissue lysates were separated with SDS-PAGE gels and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Eschborn, Germany). The blots were blocked in 5% milk for 1 h at room temperature. PVDF membranes were incubated with the primary antibodies overnight in a cold room at 4 °C. Subsequently, bound primary antibodies were reacted with corresponding secondary antibodies for 1 h at room temperature and evaluated with by chemiluminescence.
Plasmids, lentiviral infection, and transfection
Human circRNA_0008532 and MTGR1 cDNA was amplified by PCR and cloned into a lentiviral vector (GeneChem, Shanghai, China). Oligos of circ_0008532 and MTGR1 shRNAs were synthesized and inserted into a lentiviral vector (GeneChem, Shanghai, China). Stable cell lines were selected for 10 days with 0.5 mg/ml puromycin. The miR-155-5p\miR-330-5p mimics, negative control, and anti-miR-155-5p\anti-miR-330-5p inhibitor were purchased from RiboBio (Guangzhou, China). MiRNA or miRNA inhibitor was transfected with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions.
Migration and invasion assays
The capacity for cell migration and invasion was evaluated by using Transwell chambers (Corning Life Sciences, MA, USA). After pretreatment, 4 × 104 cells suspended in 200 μL of serum-free medium were seeded into the upper chamber of the Transwell system, and medium supplemented with 10% FBS was added to the lower chamber. For invasion assays, cells were seeded in pre-coated Matrigel Transwell insert chambers. After incubation for 24 h, cells remaining on the top surface were removed, and cells migrated to the lower surface of the membrane were fixed and stained with 0.1% crystal violet.
Wound healing assay
Cells were seeded in 12-well plates and grown under permissive conditions until 90% confluence was reached. A linear wound was created in the confluent monolayer using a pipette tip, and cells were incubated for 24 h in serum-free medium in a temperature-and CO2-controlled incubator. Wound closure was evaluated by measuring the distance between opposite edges of the wound.
One hundred μl of precooled Matrigel (Becton, Dickinson and Company, NJ, USA) was coated into each well of a 24-well plate and polymerized for half an hour at 37 °C. HUVECs (1 × 105) with 500 μl medium from different groups were added to each well and incubated at 37 °C under 5% CO2. Wells were evaluated every 2 h. At the proper time, capillary tube structures were photographed under a bright-field microscope, and quantified by measuring the total length of the completed tubes.
RNA fluorescence in situ hybridization (RNA-FISH)
Cy3-labeled circ_0008532 and Dig-labeled locked nucleic acid miR-155-5p and miR-330-5p probes were purchased from RiboBio (Guangzhou, China). Images were obtained using a Fluorescent in Situ Hybridization kit (RiboBio, Guangzhou, China) following the manufacturer’s instructions. All data were analyzed with a Nikon A1Si Laser Scanning Confocal Microscope (Nikon Instruments Inc., Japan).
RNase R digestion
Total RNA (2 μg) was incubated for 20 min at 37 °C with or without 3 U/μg of RNase R. The resulting RNA was purified using an RNeasy MinElute Cleanup Kit (Qiagen, Now York, USA).
RNA binding protein immunoprecipitation assay (RIP)
The RNA binding protein immunoprecipitation (RIP) assay was performed using the Magna RIP Kit (Millipore, USA) and Ago2 antibody (Cell Signaling Technology, USA) in accordance with the manufacturer’ instructions. In brief, 107 transfected cells were washed in ice-cold PBS twice, lysed in an equal volume of RIP lysis buffer and incubated with 2 μg of primary antibodies for 2 h at 4 °C. Fifty μ of prepared magnetic beads were subsequently added to each sample and incubated at 4 °C overnight. Beads were washed briefly with RIP buffer five times and resuspended in Trizol (Invitrogen). The binding products were detected with real-time qPCR.
All animal experiments were carried out in accordance with NIH Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Care Committee of Tongji Medical College. BALB/c-nu mice (3–4 wks of age) were purchased from the Center of Experimental Animals of Tongji Medical College at Huazhong University of Science and Technology, and were randomly divided into groups (n = 5/group). 2 × 106 cells were injected into the tail-vein of each mouse to establish a metastasis model. On day 49, all animals were anesthetized with xylazine (10 mg/kg) and were then euthanized by cervical dislocation. Lungs were excised and subjected to pathologic examination. The In Vivo Optical Imaging System (In Vivo FX PRO, Bruker Corporation) was used to acquire fluorescent images of xenografts in nude mice.
Statistical analysis
Statistical analysis were conducted with SPSS 16.0 software and significance was analyzed with the Student’s t-test. The cut-point of MTGR1 was defined as the median. Overall survival and recurrence-free survival curves were calculated with the Kaplan–Meier method and compared using the log-rank test. In this study, P < 0.05 was considered statistically significant. Data from at least 3 independent experiments are expressed as mean ± SD.
Discussion
Muscle invasive bladder cancer (BC) is a deadly disease. Currently, the therapeutic approach to BC is based primarily on surgery and standard chemotherapy, and novel therapeutic strategies are urgently needed for the treatment of metastatic BC [
13]. In recent years, the role of dysregulated non-coding RNAs (ncRNAs) in the proliferation, migration, invasion, and angiogenesis of cancer cells have generated significant scientific interest. The non-coding part of the genome accounts for more than 90% of the human genome. Studies have demonstrated that ncRNAs play critical roles in tumorigenesis and pathologic processes in many human cancers [
14,
15]. CircRNA, as abundant stable ncRNAs, has been demonstrated to play a prominent upstream role in BC development. For example, cTFRC is upregulated in BC, and is involved in invasion and proliferation in BC cell lines [
16]. Moreover, circRNA hsa_circ_0068871 regulates FGFR3 expression and activates STAT3 by targeting miR-181a-5p to promote bladder cancer progression, showing some of the important roles of circRNA in BC [
17].
In the current study, we explored the effect of the novel circRNA circ_0008532 on the aggressiveness of BC and identified the regulatory mechanism of miR-155-5p/miR-330-5p/MTGR1 signaling. Our results indicate that elevated expression of circ_0008532 increases the invasive capacity of BC cells. Circ_0008532 functions as a molecular sponge for miR-155-5p and miR-330-5p and weakens the inhibitory effect of these molecules on the downstream target gene MTGR1. These results suggest that Circ_0008532 has the potential to regulate the migration, invasion, and angiogenesis of BC cells, which in turn promote BC progression.
It is well-known that circRNAs may act as miRNA sponges to regulate the expression of target genes [
18]. For example, Circ-EZH2 promotes cell growth, migration and invasion but inhibits cell apoptosis through miR-1265 Sponge Activity in Glioma [
19]. CircARHGAP10 suppresses cell proliferation and metastasis in non-small-cell lung cancer by acting as a miR-150-5p sponge which promotes GLUT1 expression [
20]. Here we found that circ_0008532 enhances the aggressiveness of BC mainly through sponging miR-155-5p and miR-330-5p. Circ_0008532 is located mainly in the cytoplasm, which is regarded as a major characteristic of miRNA sponges. Second, the circ_0008532 expression level is negatively correlated with miRNAs expression. Third, bioinformatics prediction and luciferase reporter assays show that circ_0008532 and the MTGR1 3′ UTR share identical miR-155-5p/miR-330-5p response elements and may therefore bind competitively to miR-155-5p/miR-330-5p. This study thus reveals that a circ_0008532/miR-155-5p/miR-330-5p/MTGR1 axis exists in BC.
MTGR1, also known as CBFA2T2, is a member of the Myeloid Translocation Gene (MTG) family, and this family of molecules are transcriptional corepressors lacking both enzymatic activity and DNA binding capabilities [
21]. The MTGR1 gene is located at chromosome 20q11.21 and has been found to be involved in the regulation of the nuclear corepressor/histone deacetylase complex in hematopoietic differentiation [
22]. In addition, MTGR1 has been shown to contribute to Notch signaling inhibition and regulation of intestinal lineage allocation [
23]. Evidence has demonstrated that MTGR1 is involved in the development of cancer. Barrett et al. reported that MTGR1 is required for efficient inflammatory carcinogenesis in the murine AOM/DSS colitis-associated carcinoma model [
24]. Parang et al. showed that MTGR1 is downregulated in human colorectal cancer and has a context-dependent effect on intestinal tumorigenesis [
25]. However, the biologic functions of MTGR1 in bladder cancer have not been investigated. Our study shows that MTGR1 is increased in bladder cancer tissues, and that overexpression of MTGR1 leads to enhanced progression in bladder cancer. Furthermore, circ_0008532 promotes progression of bladder cancer by promoting MTGR1 expression and, in turn, inhibits the activity of Notch signaling. Our findings are supported by the study of Rampias and colleagues, who showed that genetic inactivation of Notch signaling leads to tumorigenesis in urothelial cancer [
26]. Similarly, Maraver et al. demonstrated that Notch serves as a tumor suppressor in the bladder and that loss of this pathway promotes mesenchymal changes and invasive features [
27]. Together, these findings reveal a crucial link between MTGR1 and the Notch signaling pathway during bladder cancer development and progression.
Conclusions
In conclusion, the current study shows circ_0008532 may be required for BC progression, and this molecule has potential as a clinical biomarker for BC. Moreover, circ_0008532 serves as a sponge for miR-155-5p/miR-330-5p to reduce the inhibitory effect on MTGR1, and thus enhances the expression of MTGR1 and inhibits the activity of the downstream Notch signaling pathway.
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