Background
Bladder cancer (BC) is the most common urinary system malignancy with an estimated 81,400 new cases in 2019 in the United States [
1]. For patients with advanced-stage or chemotherapy-refractory BC, the prognosis remains poor in despite of improvements in surgical techniques and medical therapy [
2,
3]. The development of BC is a complex process and epigenetic abnormalities have been demonstrated to play critical roles in BC pathogenesis [
4,
5], including DNA and histone modifications, chromatin remodeling, RNA methylation and so on. In particular,
N6-methyladenosine (m
6A), the most prevalent RNA methylation, is emerging as critical regulator in multiple fundamental biological processes [
6,
7]. However, our understanding of the regulatory roles and the underlying mechanisms for m
6A in BC is still limited.
Circular RNAs (circRNAs) are a novel class of single-stranded RNAs and characterized by covalently closed continuous loops and resistance to RNase R digestion [
8]. Increasing RNA-sequencing analyses have revealed evolutionary conservation and abundance of circular RNAs, suggesting specific roles of circRNAs in cellular physiology [
9,
10]. Specifically, circRNAs have been verified as “microRNA (miRNA) sponges,” harboring multiple miRNAs and functioning as miRNA inhibitors [
11]. For example, our previous research demonstrated that BCRC-3 suppressed proliferation of BC cells through miR-182-5p/p27 axis [
12]. Nevertheless, genome-wide studies have demonstrated that miRNA sponging activity cannot be generally applied, and other mechanisms have also been proposed, such as acting as platforms for protein interaction, translating into peptides or proteins [
13,
14]. For example, overexpressed circSTAG1 captured ALKBH5 and decreased the translocation of ALKBH5 into the nucleus, leading to increased m
6A methylation of fatty acid amide hydrolase (FAAH) messenger RNA [
15]. Accumulating evidences show that circRNAs are frequently deregulated in various human cancers and participate in multiple biological processes [
16]. However, the roles and mechanisms of circRNAs in the process of recognitions of m
6A methylation remain largely elusive.
The insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1), also known as IMP1, CRD-BP, ZBP1, or VICKZ1, belongs to a conserved family of RNA-binding, oncofetal proteins (IGF2BP1–3). Recent studies indicate that IGF2BP1 has the most conserved ‘oncogenic’ role of the IGF2BP family in tumor-derived cells, by affecting RNA stability, translatability, or localization [
17]. Crosslinking immunoprecipitation (CLIP) analyses showed that IGF2BPs preferentially recognize m
6A-modified mRNAs and facilitate the stability and translation of potential mRNA targets in an m
6A-dependent manner, therefore having impacts on gene expression output [
18,
19]. Recently, non-coding RNAs (ncRNAs) have been demonstrated to be involved in modulating the expression and function of IGF2BP1. For example, the post-transcriptional control of IGF2BP1 expression by let-7 microRNAs is suggested to modulate tumor cell fate [
20]. A conserved direct interaction of the lncRNA THOR with IGF2BP1 showed that THOR contributes to the mRNA stabilization activities of IGF2BP1 [
21]. However, it remains poorly understood the involvement of IGF2BP1 in BC development and how it might be modulated by circular RNA.
In this study, we revealed the oncogenic functions of IGF2BP1 in BC progression and identified a number of novel circular RNAs interacting with IGF2BP1 through high-throughput sequencing. A circRNA derived from PTPRA pre-mRNA (circPTPRA) was screened out. It showed that circPTPRA could suppress the growth and aggressiveness of BC cells by competitively binding with KH domains of IGF2BP1 and blocking its interaction with downstream target m6A-modified mRNA, MYC and FSCN1. The results of this study delineate novel mechanisms of circRNA/IGF2BP1-mediated regulation of tumor progression and provide opportunities for therapeutic intervention in BC.
Methods
Human tissue specimens
A total of 64 pairs of BC tissues and corresponding adjacent noncancerous bladder epithelial tissues were obtained from patients who underwent radical cystectomy in Union Hospital of Tongji Medical College of Huazhong University of Science and Technology (Wuhan, China), from 2015 to 2018. All the specimens were confirmed by at least two experienced histopathologists independently according to the criteria of the sixth edition TNM classification of the International Union Against Cancer. All specimens were snap-frozen in liquid nitrogen upon collection and stored at − 80 °C until use. Clinical information of the patients with BC was listed in Table S
1. The study was approved by the Tongji Medical College of Huazhong University of Science and Technology Research Ethics Committee, and each patient signed informed consent before the research started.
Cell culture
BC cell line (EJ) was purchased from American Type Culture Collection (ATCC, USA). The human metastatic bladder cancer cell line T24T, a lineage-related lung metastatic variant of invasive bladder cancer cell line T24, was obtained from the Departments of Urology, University of Virginia (Charlottesville, VA) as a gift in 2010 and was subjected to DNA tests and authenticated in our previous studies. T24T was cultured in DMEM (Invitrogen, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA). EJ was cultured in RPMI1640 (Gibco, USA) supplemented with 10% FBS, 1% penicillin/streptomycin. All cell lines were confirmed 3 months before the beginning of the study based on a short tandem repeat method and were tested negative for mycoplasma contamination.
RT–PCR and real-time quantitative RT–PCR
Total RNA was isolated from tissues and cell lines with RNeasy Mini kit (QIAGEN, Germany) according to the manufacturer’s instructions. RNA was reverse transcribed using the PrimeScript RT Master Mix (Takara, Japan). Real-time PCR was performed using SYBR Premix Ex TaqTM kit (Takara, Japan) and primers (Table S
2). The results were analyzed with the Step OnePlus Real Time PCR System (Applied Biosystems, USA). The 2
-△△Ct method was used to quantify the transcript levels.
CRISPR/Cas9 KO
T24T and EJ cells were transiently transfected with the lentiCRISPR v2 plasmid (Addgene Plasmid #52961) containing IGF2BP1 single-guide RNA (sgRNA) using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Single-cell colonies were selected, and knockout efficiency was tested by Western blot analysis and Sanger sequencing. For Sanger sequencing, genomic DNA was extracted from sgCtrl and sgIGF2BP1#1 cells. PCR was performed to amplify the region flanked by the target site. The sgRNA sequences used are listed below: sgIGF2BP1#1, TATTCCACCCCAGCTCCGAT; sgIGF2BP1#2, GAGCGTGACCCCCGCGGACT.
Western blot
Tissue or cellular protein was extracted with RIPA Lysis Buffer (Thermo Scientific, USA) according to the instructions. The concentration of total protein was measured by bicinchoninic acid (BC) protein assay kit (Beyotime, China). Western blot was conducted as previously described, with antibodies specific for IGF2BP1 (ab82968), MYC (ab32072), FSCN1 (ab126772), HSPA6 (ab69408), FLAG (ab1162), GAPDH (ab8245), and β-actin (ab8226).
Immunohistochemistry
Immunohistochemistry was performed as previously described, with antibodies specific for Ki-67 (Proteintech, 1:200) or CD31 (Proteintech, 1:200). Images were captured by an Olympus FSX100 microscope (Olympus, Japan). Protein expression levels were analyzed by image-pro Plus 6.0. software through calculating the integrated optical density per stained area (IOD/area).
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 Tong ji Medical College (approval number: 20192290). For in vivo tumor growth studies, 1 × 106 treated T24T cells were subcutaneously injected into the right axilla of blindly randomized four-week-old female BALB/c nude mice (n = 5 per group). Four weeks after injection, the mice were sacrificed. Tumor growth rates were monitored every other day, and tumor volume was calculated according to the formula (Tumor volume = π/6 × length×width2). For in vivo metastasis studies, 2 × 106 treated T24T cells were injected into each blindly randomized 4-week-old BALB/c nude mice (n = 5 per group) through the tail vein. Ten weeks after injection, the mice were sacrificed. The survival time of each mouse was monitored and recorded. The In-Vivo FX PRO small animal imaging system (BRUKER Corporation, USA) was used to obtain fluorescence images of xenografts in nude mice.
RNA fluorescence in situ hybridization
RNA Fluorescence in situ hybridization was performed according to the manufacturer’s instructions. Cy3-labeled circPTPRA probes were designed and synthesized by RiboBio (Guangzhou, China). The signals of circPTPRA were detected by Fluorescent In Situ Hybridization Kit (RiboBio, China). The images were captured using Nikon A1Si Laser Scanning Confocal Microscope (Nikon Instruments Inc., Japan).
Fluorescence immunocytochemical staining
BC cells were grown on coverslips, and treated with antibodies specific for IGF2BP1 (8482S, CST; 1:100 dilution) at 4 °C overnight. Then, coverslips were treated with Alexa Fluor 488 goat anti-rabbit IgG (1:400 dilution) and DAPI (300 nmol/L) staining. The images were photographed under a Nikon A1Si Laser Scanning Confocal Microscope (Nikon Instruments Inc., Japan).
Cell cycle assay
Cell cycle analysis was performed by flow cytometry. Cells were harvested and fixed in 75% ice-cold ethanol at 4 °C overnight. The fixed cells were washed with PBS twice and then stained with propidium iodide (PI) buffer (BD Pharmingen, USA). Then, cell cycle analysis was performed by FACS scan flow cytometer. ModFit LT 2.0 was used to analyze the data.
In vitro cell migration and invasion assays
The abilities of cell migration and invasion were measured using transwell chambers (corning, USA) with 8 μm pore filters according to the manufacturer’s instructions. Cells were suspended in 200 μl serum-free medium (5 × 104 cells per well for migration, and 1 × 105 per well for invasion) and were added to the upper chambers coated with or without 50 μl of Matrigel (BD Biosciences, USA). DMEM medium containing 20% FBS was added to the bottom of chambers. After incubation at 37 °C for 24 h (migration assay) or 48 h (invasion assay), cells on the upper surface were removed with a cotton swab. Cells that migrated or invaded into the bottom of the membrane were fixed with 4% paraformaldehyde, stained with crystal violet solution, and then visualized under a microscope. The cell numbers were counted in five random fields of view.
RNA pull-down assays
The biotinylated probe of circPTPRA was designed and synthesized by RiboBio (Guangzhou, China). The sequence of circPTPRA probe was listed in Table S
2. Pull-down assay was performed as described in our previously studies. Briefly, M-280 streptavidin magnetic beads (Invitrogen, USA) at 25 °C for 2-4 h, and then total cell lysates with Protease/Phosphatase Inhibitor Cocktail and RNase inhibitor added were incubated with circPTPRA probe or oligo probe at 4 °C overnight. After washing thoroughly three times, the RNA complexes bound to the beads were eluted and extracted with RNeasy Mini Kit (QIAGEN) and were analyzed by qRT-PCR, and the RNA–protein binding mixture was boiled in SDS buffer and the eluted proteins were detected by western blot.
RNA stability assay for mRNA lifetime
T24T cells with stably overexpressed circPTPRA were seeded into 6-well plates to get 50% confluency after 24 h. Cells were treated with 5 μg/ml actinomycin D and collected at indicated time points. The total RNA was extracted by miRNeasy Kit (Qiagen) and analyzed by RT-PCR. The turnover rate and half-life of mRNA was estimated according to previously published paper [
18,
22]. The primers for MYC and FSCN1 are listed in Table. S
2.
RNA immunoprecipitation (RIP)
Cells seeded in a 15-cm dish at 70–80% confluency were cross-linked by ultraviolet light at 254 nm (200 J/cm2), then harvested and lysated. RNA immunoprecipitation (RIP) assay was performed according to the instructions of the Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore, USA), using antibody specific for IGF2BP1 (8482S, CST), FLAG (ab1162, abcam) or a corresponding control IgG (mouse IgG (CS200621, Millipore) for FLAG, rabbit IgG (NI01, Millipore) for IGF2BP1. Input and co-immunoprecipitated RNAs were extracted with a RNeasy Mini kit (QIAGEN, Germany) according to the manufacturer’s instructions and analyzed by qRT-PCR or RNA-seq.
RNA sequencing
Total RNA was isolated from circPTPRA-overexpressed T24T and EJ cells and the corresponding control cells using RNeasy Mini kit (Qiagen). Transcriptome sequencing on an Illumina HiSeq X Ten platform was carried out by SeqHealth Tech (Wuhan, China).
Dual-luciferase reporter assay
DNA fragments of wild-type and mutant CRD were synthesized and cloned into the pMIR-REPORT vector (Promega, USA) to constructed CRD firefly luciferase reporters.
HEK293T cells were seeded in 24-well plates at 60–80% confluency before transfection. The CRD firefly luciferase reporter plasmids (pMIR-CRD-WT and pMIR-CRD-mut, respectively) and renilla luciferase reporter control vectors (pRL-TK) were co-transfected with circPTPRA plasmid or vectors to examine the CRD luciferase activities. On the other hand, the pMIR-CRD-wide type reporter plasmids were co-transfected with circPTPRA plasmids and IGF2BP1 expression plasmids using Lipofactamine 2000 (Invitrogen). The firefly and renilla luciferase activities were measured after 48 h with Dual-Luciferase® Reporter Assay System (Promega, USA) according to the manufacturer’s protocol.
Statistical analysis
All the data statistical analyses were performed using GraphPad Prism 7.0 software (La Jolla, USA) to assess the differences between groups. Data were shown as mean ± SEM. The chi-squared test was used to assess the association of the expression of IGF2BP1 or circPTPRA with the patient’s clinicopathologic characteristics. Kaplan–Meier survival curve was employed to depict the OS distributions and Log-rank test was used to assess survival difference. Independent sample t test was employed to assess statistical significance of comparisons between groups. Pearson’s correlation coefficient assay was used to analyze the expression correlation. One-way analysis of variance was performed to evaluate the group difference. P < 0.05 was considered statistically significant.
Discussion
Recent studies have identified IGF2BPs as novel carcinogenesis factors in a number of solid tumors, including ovarian, breast, melanoma and hepatocellular tumors, and its high expression is associated with metastasis and poor prognosis [
25]. Whereas the oncogenic role IGF2BP1 and its paralogs in BC remains unknown. In this study, our results indicated that high expression of IGF2BP1 was associated with poor prognosis in BC. As a RNA-binding protein, IGF2BP1 apparently ‘cages’ their target mRNAs in cytoplasmic protein–RNA complexes, preventing the premature decay of specific target transcripts in an RNA-dependent manner [
26]. The stable ‘caging’ of transported mRNAs allows for their ‘long-distance’ transport during cellular stress as well as transient storage [
27]. In this study, our RIP-Seq data revealed that IGF2BP1-binding sites were enrichment in protein-coding transcripts region (CDS) and m
6A-dependent binding mode was on top of the primary sequence. Our gain- and loss-of-function studies indicated that IGF2BP1 facilitated the proliferation, migration and invasion of cancer cells, suggesting the oncogenic roles of IGF2BP1 in BC. These findings indicate that IGF2BP1 could be served as therapeutic target, as well as possibly be used for potential clinical diagnosis and prognosis evaluation of BC.
The most prevalent RNA methylation,
N6-methyladenosine (m
6A), occurs in approximately 25% of transcripts at the genome-wide level and is enriched in 5′- and 3′ -untranslated regions. m
6A is installed by m
6A methyltransferases (METTL3/14, WTAP), reverted by m
6A demethylases (FTO, ALKBH5) and recognized by reader proteins (YTHDF1/2/3, IGF2BPs) [
28‐
30]. Accumulating evidences show that, m
6A RNA methylation has an outsize effect on RNA production/metabolism and participates in the pathogenesis of multiple diseases including cancers [
31]. As a key m
6A reader, the transcription of IGF2BP1 is modulated by negative as well as positive feed-back regulation of proteins, including b-catenin (CTNNB1) and MYC [
32,
33]. The up-regulated three lncRNAs (HCG11, GHET1, and THOR) can elevate IGF2BP1 level, potentially via forming the surprisingly long half-life of IGF2BP-RNA complexes [
21,
34,
35]. Besides, LncRNA LIN28B-AS1 could alter the LIN28B mRNA stability by physical combination with IGF2BP1 [
36]. In this study, the oncogenic function of IGF2BP1 in BC growth and aggressiveness was reversed by ectopic expression of circPTPRA in vivo and in vitro through reducing its interaction with downstream target m
6A-modified mRNA, while the expression level of IGF2BP1 was not affected. These results extend our knowledge about the regulation mode of IGF2BP1 function in cancer cells, which is mediated by the direct binding of circRNA with IGF2BP1. Enhancing the effect of tumor suppressive circRNAs, such as circPTPRA, may act as efficient therapeutic strategies for future cancer therapy. Exogenous circRNAs could be achieved by gene therapy where DNA cassettes designed for circRNAs expression are delivered, or by transfection of purified, in vitro-generated circRNAs [
37]. These stably transfected circRNAs produce more quantity of proteins than modified linear mRNA or unmodified counterparts.
IGF2BP1 consists of six canonical RNA-binding domains, including two RNA-recognition motifs (RRMs) in the
N-terminal part and four hnRNP-K homology (KH) domains in the
C-terminal region [
38]. In vitro studies revealed that the stabilization of IGF2BP1-RNA complexes is mainly facilitated via the KH3/4 domain, which could potentially contribute to the binding of IGF2BP1 to the MYC-CRD (coding region stability determinant) RNA [
18,
39]. In this study, we found that KH3/4 domain of IGF2BP1 was also necessary for its interaction with circPTPRA, and circPTPRA promoted the endonuclease-directed decay of downstream mRNAs via forming a circRNA/IGF2BP1 complex to sequester the transcripts. This mode of regulation is presumably relied on the affinity of the association between circRNA and IGF2BP1, resulting in the competitive interaction of circRNA and target mRNAs with IGF2BP1. It has been previously reported that circNSUN2 could enhance the stability of HMGA2 mRNA to promote colorectal carcinoma metastasis progression by forming a circNSUN2/IGF2BP2/HMGA2 RNA-protein ternary complex in the cytoplasm [
40]. Here, we revealed a different role of circRNA in regulation of IGF2BP1 function, which acts as an effective endogenous blocker.
Silencing of IGF2BP1 globally down-regulates target gene expression in mRNA level, including FSCN1, TK1, MARCKSL1, and MYC [
18]. Recent studies have shown an association between the up-regulated expression of FSCN1 and increased invasiveness of carcinomas in the urinary bladder, which suggests that FSCN1 may be a marker of aggressive bladder cancer [
41,
42]. The expression of FSCN1 could be indirectly regulated by lncRNA through “miRNA sponge” effect at the transcriptional level, including lncRNA-UCA1, LINC00152 and ZEB1-AS1 [
43‐
45]. Oncogene MYC is known to be aberrantly expressed in BC and acts as a master regulator of genes involved in cell cycle progression, cell growth, differentiation, metabolism, and apoptosis [
46]. Mechanisms of MYC deregulation in BC include signal transduction transcriptional regulation [
47], miRNA mediated post-transcriptional regulation [
48] and DNA mutation [
49]. Besides, alteration of m
6A levels also participates in cancer pathogenesis via regulating expression of MYC. For example, depletion of METTL3 in BC cells decreased the stability of MYC transcripts through affecting m
6A abundance mainly around the stop codon and 3′-UTR regions [
50].
N6-methyladenosine modification in the CRD of the MYC mRNA enhances the association of IGF2BPs and interferes with the endonuclease-directed decay of the MYC mRNA [
51]. In the current study, we found that FSCN1 and MYC, but not other targets, were involved in circPTPRA/IGF2BP1 regulation of m
6A recognition and mRNA stability. Our observations indicate that the downstream effectors of IGF2BPs recognition of m
6A have cell- and tissue- specificities, and the targets of each circRNA and IGF2BPs complex need to be individually identified.
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