Background
Bladder cancer is overall the ninth most common malignancy and the most prevalent cancer in urinary system, with an estimated 81,180 new cases and 17,100 deaths in 2022 in the United States [
1,
2]. Approximately 25% of new patients are diagnosed as muscle-invasive or metastatic disease [
3]. The current gold standard for the diagnosis of BCa involves the use of cystoscopy and cytology. Recently, a new non-invasive urine tumor DNA methylation detection could improve the diagnostic rate of early tumors [
4]. Transurethral resection of bladder tumors and radical cystectomy are recommended for early-stage patients, but when the tumors deteriorated to advanced stages, the cancers are no longer curable by surgical treatments [
5]. Clinically, cisplatin (CDDP)-based chemotherapy has become the standard frontline treatment for patients with advanced BCa. However, patients always obtain drug resistance after several cycles of CDDP-based treatment, which limiting overall clinical efficacy [
6,
7]. Under these circumstances, a better comprehension of the molecular mediators and regulatory mechanisms underlying CDDP resistance is of great clinical importance for BCa patients.
Circular RNAs (circRNAs), a category of noncoding RNAs, are derived from back-splicing of precursor mRNAs, with a covalently closed loop structures without 5’caps and 3’polyadenylated tails [
8,
9]. In recent years, large amounts of circRNAs have been successfully discovered and identified in a tissue-specific or cell type-specific manner by high-throughput sequencing analysis [
10,
11]. Accumulating evidence indicates that circRNAs play important roles in multiple biological functions, such as cell proliferation, migration, invasion and chemosensitivity [
12‐
14]. Our previous studies demonstrate that some dysregulated circRNAs play a vital role in chemotherapy sensitivity of BCa [
15,
16]. It has been reported that circRNAs may regulate gene expression by sponging miRNAs in a complementary base-pairing manner [
17]. In addition, circRNAs can also interact with RNA-binding proteins (RBPs) to form specific complexes that subsequently mediate the function of associated proteins [
18]. Increasing evidence indicates that circRNAs could also exert their functions through regulating transcription and encoding peptide [
19,
20]. Notably, recent studies have depicted an emerging expression profile of circRNAs in BCa, with tumor-promoting or tumor-inhibiting properties [
21,
22]. However, the functions and regulatory mechanisms of circRNAs in modulating CDDP resistance in BCa still remain largely enigmatic and need to be further explored.
In the present study, we identified a circRNA derived from the STX6 gene, hsa_circ_0007905 designated as circSTX6, by analyzing expression profiles of circRNAs in BCa. CircSTX6 was frequently upregulated in BCa tissues compared with paired noncancerous adjacent tissues, and increase of eukaryotic initiation factor 4A3 (EIF4A3) contributed to the upregulation of circSTX6. Subsequent studies showed that circSTX6 could directly sponge miR-515-3p to upregulate SUZ12 expression and consequently promote the metastasis of BCa in vitro and in vivo. Importantly, circSTX6 increased the stability of SUZ12 mRNA by strengthening its interaction with poly(A) binding protein cytoplasmic 1 (PABPC1), leading to enhanced expression of SUZ12. Notably, knockdown of circSTX6 could promote the sensitivity of BCa to CDDP chemotherapy in vitro and in vivo. Taken together, our study delineated an important role of circSTX6 in tumor progression and CDDP resistance, providing a potential therapeutic target for overcoming metastasis and chemoresistance in BCa.
RNA pulldown assay
Pull-down assay was performed as described in our previously studies [
15]. In brief, about 10
7 cells were washed in ice-cold PBS, lysed in 500 µl Co-IP buffer (Thermo Scientific, USA) supplemented with a cocktail, PMSF, and RNase inhibitor (Solarbio, China), and then incubated with 3 µg biotinylated DNA
circSTX6 sense probe or antisense probe for 2 h at room temperature. Then the reactions were mixed with 50 µl Streptavidin C1 magnetic beads (Invitrogen, USA) for another hour, which were prewashed twice with tris buffer. The beads were washed briefly with Co-IP buffer for five times. Finally, the retrieved RNA was used for qRT-PCR analysis. Biotinylated
circSTX6 sense probes or antisense probes (Supplementary Table
2) were synthesized by RiboBio (Guangzhou, China). The RNA-protein binding mixture was boiled in SDS buffer and the released proteins were detected by western blot and silver staining. Mass spectrometry was conducted at Shanghai Luming biological technology co.,LTD (Supplementary Table
3).
Biotin-labeled miRNA capture
Stably overexpressed circSTX6 BCa cells were transfected with biotinylated wild-type or mutant miR-515-3p using Lipofectamine RNAiMax. After 48 h of transfection, the cells were harvested, sonicated and incubated with Streptavidin C1 magnetic beads (Invitrogen, USA) at 4 °C on the rotator overnight. The bound RNAs were purified using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, China) and the abundance of circSTX6 in bound fractions was tested by qRT-PCR.
Western blot analysis
Protein extracts were isolated from BCa cells with RIPA lysis buffer containing protease inhibitor cocktail and PMSF. An equal amount of total protein lysates was separated by SDS-PAGE gel and transferred onto PVDF membranes. The membranes were incubated with primary antibodies against Actin (BE0021, 1:5,000, bioeasytech), EIF4A3 (catalog no. 17504-1-AP, 1:1,000, Proteintech), SUZ12 (catalog no. 20366-1-AP, 1:1,000, Proteintech), PABPC1 (catalog no. 10970-1-AP, 1:1,500, Proteintech) overnight at 4 °C, followed by an incubation with HRP-conjugated secondary goat anti-mouse (catalog no. SA00001-1, 1:4,000, Proteintech) or goat anti-rabbit (catalog no. SA00001-2, 1:4,000, Proteintech) for 1 h at room temperature. Finally, the protein bands were visualized using ECL substrate kit via QuickChemi 5200 Imaging System.
RNA immunoprecipitation
Briefly, the proteinA/G magnetic beads (MCE, USA) were incubated with anti-EIF4A3 antibodies (Proteintech, USA), anti-PABPC1 antibodies (Proteintech, USA) or IgG negative control antibody (Proteintech, USA). Subsequently, BCa cells were lysed and incubated with the corresponding antibody-coated beads. Co-precipitated RNAs were then purified and measured by qRT-PCR for enrichment. The primer sequences were listed in Supplementary Table
2.
Wound healing, migration and invasion assays
For wound healing assay, straight scratch was made with a sterile 200 µL pipette tips (0 h) in the six-well plates and photographed immediately, and then the cells were cultured with serum-free medium. After 24 h, images of wounds were captured, and the distance was measured and normalized to the 0 h control as the relative migration rate.
For transwell migration and invasion assays, a 24-well transwell chamber (Costar, USA) with or without Matrigel (BD Science, USA) was used to detect cell invasive and migratory abilities, respectively. Cells were suspended in 200 µL serum-free medium and added to the upper chambers (5 × 104 cells per well for migration, and 1 × 105 per well for invasion). About 600 µL of medium supplemented with 10% FBS was applied to the lower chambers. After incubation for 24 or 48 h, cells in the upper chamber were softly removed with cotton swabs, and cells that migrated to the lower membrane surface were fixed with 4% paraformaldehyde and stained with crystal violet in PBS for photographing and counting. The migrated and invaded cells were counted in three randomly selected fields.
Self-renewal of cancer stem cells was assessed by tumor sphere formation. Briefly, cells were plated in ultra-low adhesion 12-well plates at a density of 1000 cells/well and grown in a serum-free DMEM/F12 medium (Procell, China) containing 20 ng/ml of epidermal growth factor, 5 µg/ml of insulin, 0.4% bovine serum albumin, 2% B27 and 1% penicillin/streptomycin. After 10 days in culture, the number of spheres was quantified by counting spheres under a phase contrast microscope (Olympus, Japan).
EdU assay
EdU assay was performed using Cell-Light™ EdU ApolloR567 In Vitro Kit (RiboBio, China) according to manufacturer’s protocols. Briefly, BCa cells were seeded in 96-well plate and labeled with 50 µM EdU for an additional 2 h. Then, the cells were fixed with 4% paraformaldehyde in PBS and stained. Finally, the cells were subjected to nuclear staining with DAPI for 15 min at RT. The images were captured with an Olympus FSX100 microscope (Olympus, Japan) and cell proliferation rate was calculated.
Cell viability analysis
The BCa cells were seeded in 96-well plate with 5000 cells per well, and were cultured at 37˚C overnight. Then, the cells were treated with a series of dilute concentrations of cisplatin for 24 h. To evaluate the cytotoxicity of cisplatin, cell viability was tested by CCK-8 kit (Dojindo, Japan) according to the manufacturer’s instructions.
The EJ and UMUC3 cells stably transfected with circSTX6 shRNA#1 and corresponding control were harvested, and then we extracted their total RNA. A high-quality RNA sample was used to construct a sequencing library in Majorbio Technologies (Shanghai, China). The results of RNA-seq were listed in Supplementary Table
4. The databases of CircInteractome and Circbank were used to predict the potential miRNAs bound with circSTX6. The potential target genes mediated by miR-515-3p were acquired from TargetScan, miRDB and miRTarBase. In addition, RBPDB and RBPmap were used to predict the RBPs interacted with circSTX6.
Fluorescence in situ hybridization (FISH) and immunofluorescence (IF)
Cy3-labeled probes targeting circSTX6 and FAM-labeled miR-515-3p probes were designed and synthesized by RiboBio Technology Co. Ltd. The signals of the probes were detected by the FISH Kit (RiboBio, China) according to the manufacturer’s instructions. Briefly, BCa cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After prehybridization, cells were hybridized at 37 °C overnight in a dark, humid chamber. Nuclei were counterstained with DAPI. For IF, cells were fixed, permeabilized, blocked with 5% BSA for 30 min at 37 °C, and then incubated with PABPC1 (catalog no. 10970-1-AP, 1:50, Proteintech) or γH2AX (#9718, 1:200, CST) primary antibody overnight at 4 °C. After washing twice with PBS and then cells were incubated with the Alexa Fluor 488 or the Alexa Fluor 594 (ABclonal, China) for 1 h at 37 °C, followed sealing with parafilm containing DAPI. Fluorescent images were acquired using a confocal microscope (Olympus, China).
Dual-luciferase reporter assay
BCa Cells were seeded into 24-well plates (5 × 104 cells per well). The cells were co-transfected with psiCHECK2 circSTX6 3’-UTR-wide type/mutant reporter vector and miR-515-3p to examine the miRNA binding abilities. The psiCHECK2 SUZ12 3’-UTR-wide type/mutant reporter vector was co-transfected with miR-515-3p to determine the 3’-UTR activity of SUZ12. On the other hand, the psiCHECK2 SUZ12 3’-UTR reporter vector was co-transfected with PABPC1 overexpression plasmid/shRNA plasmids or circSTX6 overexpression plasmid to determine the 3’-UTR activity of SUZ12. After transient transfection for 48 h, the firefly and Renilla luciferase activities were measured with a dual-luciferase reporter assay system (Promega, USA) according to the manufacturer′s protocol. Renilla luciferase activity was normalized to the luminescence of firefly luciferase.
RNA stability assay
RNA stability was determined using ActD treatment. In short, cells were seeded in 6-well plates. Up to 60% confluency after 24 h, cells were treated with 5 µg/ml of ActD for 0, 3, 6, 9 or 12 h before RNA isolation. The
SUZ12 mRNA levels were detected by qRT-PCR assay. The half-life of
SUZ12 mRNA was estimated according to our previously published paper [
16].
Animal experiments
All mice used in this study were housed in obedience with the institutional guidelines and approved by the Institutional Animal Care and Use Committee of the Chinese PLA General Hospital. For the in vivo tumor metastasis studies, six-week-old male BALB/c nude mice were preserved in SPF-grade animal laboratory and randomly divided into three groups. EJ cells that stably transfected with circSTX6 knockdown plasmid or scramble plasmid were injected into the nude mice via tail vein (2 × 106 cells per mouse). After several weeks, all mice were sacrificed. The In-Vivo FX PRO (Bruker, USA) was used to obtain fluorescence images of xenografts in nude mice.
For in vivo drug studies, BALB/c nude mice (6 weeks old) were randomly divided into four groups, and stably downregulated circSTX6 or control EJ cells (2 × 106 cells per mouse) were injected subcutaneously into the right flank of the nude mice. For drug treatment, mice were treated with PBS or CDDP via intraperitoneal injection three-times weekly at the dose of 1 mg/kg. Tumor volume was measured with calipers and calculated as length × width2 × 0.5. After 4 weeks, all mice were sacrificed, and the tumors were excised and weighed.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0. Data were shown as the mean ± standard deviation (SD). Kaplan-Meier method and log-rank test were used to calculate overall survival rates. The significance of intergroup differences was determined with Student’s t-test or one-way ANOVA. Correlations were analyzed by Pearson’s correlation test. P < 0.05 was considered statistically significant and was marked with an asterisk.
Discussion
Bladder cancer is the most common malignancy of urinary tract with high morbidity and mortality rates [
40], with a gradually increasing incidence these years in China [
41]. CDDP-based chemotherapy is a most predominant chemotherapeutic strategy for most cancers, including BCa. However, resistance to CDDP agents, either intrinsic or acquired, is the main cause of the unfavorable prognosis of BCa patients currently [
42]. Several circRNAs are crucial in regulating the CDDP chemotherapy sensitivity of BCa. For instance, our previous studies demonstrated that
circ0008399 could promote CDDP resistance by promoting the assembly of m
6A methyltransferase complex in BCa [
15]. Another study reported that Cdr1as could improve the CDDP chemosensitivity of BCa through the
Cdr1as/
miR-1270/APAF1 axis [
43]. Herein, we uncovered that ectopic expression of
circSTX6 promoted CDDP resistance in vitro and in vivo, whereas inhibition of
circSTX6 could re-sensitize the CDDP-resistant BCa cells to CDDP. Interfering of
circSTX6 combined with CDDP significantly increased therapeutic outcomes, representing a promising therapeutic target for chemoresistant BCa. Previous studies have indicated that several factors are associated with CDDP resistance, including ATP-binding cassette (ABC) transporters, cancer stem cells (CSCs), epithelial-mesenchymal transition (EMT) and DNA damage repair [
44,
45]. In our study, we have been confirmed that knockdown of
circSTX6 could facilitate the expression of γH2AX upon CDDP treatment. SUZ12 deletion reversed the reduction of γH2AX level induced by
circSTX6 overexpression. Therefore, we speculated that
circSTX6 could promote CDDP resistance through facilitating DNA damage repair. However, the potential mechanisms of
circSTX6-mediated DNA damage repair upon CDDP treatment still remains to be further investigated.
Bladder tumors that invade the muscle layer are referred to as muscle-invasive bladder cancer (MIBC), which have a higher propensity to spread to lymph nodes and other organs. Multiple independent studies have demonstrated that lymphatic metastasis is a key prognostic factor in bladder cancer, accompanied by a reduction of the average 5-year survival rate to merely 18.6% [
46]. Previous study has illustrated that lymphangiogenesis, which means the formation of new lymphatic vessels from the preexisting lymphatics, promotes the process of lymphatic metastasis [
47]. Therefore, uncovering the mechanisms involved in lymphatic metastasis of BCa and exploring novel therapeutic strategies targeting this condition are urgently needed. It has been reported that ETV4 promotes TANs-mediated lymphangiogenesis and lymph node metastasis of BCa by regulating TANs infiltration [
46]. In addition, HSF1-PRMT5-WDR5 axis plays key roles in the lymphatic metastasis of BCa, and targeting HSF1 by KRIBB11 is a multipotent and promising therapeutic strategy for BCa patients with lymphatic metastasis [
48]. In our study, we have confirmed that
circSTX6 could promote distant lung metastasis in BCa in vivo. However, considering that BCa often exhibits lymphatic metastasis first. Whether
circSTX6 could regulate lymphangiogenesis and lymphatic metastasis of BCa still need to be further clarified.
Increasing evidence have indicated that the biogenesis of circRNAs is regulated by specific
cis-regulatory elements and
trans-acting factors [
49]. Notably, certain RBPs also play an important role in the formation of circRNAs [
50]. EIF4A3 functions as an RBP mainly localized in the nucleus, and is reported to bind RNA and form exon junction complexes, which is widely involved in exon splicing [
51]. Recent studies have shown that EIF4A3 facilitates
circMMP9 and
circARHGAP29 expression by binding with the flank sequence of their parental pre-mRNA [
52,
53]. In this study, we confirmed that EIF4A3 combined with the upstream flanking sequences of
circSTX6 pre-mRNA, which could promote the expression of
circSTX6. Besides, Chan et al. have revealed that EIF4A3 could mediate the nuclear export of spliced RNA [
51]. For example, EIF4A3 induces the cytoplasmic export of
circARHGAP29 through binding with the back-spliced junction site and the downstream flanking sequence of
circARHGAP29 in docetaxel-resistant PCa [
53]. However, whether the high expression of
circSTX6 in cytoplasm was partially through the EIF4A3 dependent cytoplasmic export should be further investigated.
The mechanism by which circRNAs exert their functions is first described as miRNA sponges [
8].
CDR1as, a classical circRNA, harboring 63 binding elements for
miR-7, can function as miRNA sponges to regulate
miR-7 target mRNA expression. Thereafter, the functions of circRNAs as a “miRNA sponges” have been discovered to participate in many biological processes. In addition, the importance of circRNA-miRNA interactions is also reported in various human cancers, including BCa.
CircHIPK3 absorbs
miR-7 to promote the expression of proto-oncogenes in colorectal cancer [
54].
CircMTO1 suppresses hepatocellular carcinoma cell progression by sponging oncogenic
miR-9 to promote p21 expression [
55].
CircRIP2 targets
miR-1305 to promote Tgf-β2/smad3 pathway, while
circITCH sponges
miR-17 and
miR-224 to enhance p21 and PTEN expression in BCa [
56,
57]. Herein, we demonstrated that
circSTX6 absorbed
miR-515-3p, and in turn promoted the expression of SUZ12. Nevertheless, the roles of
miR-515-3p are not yet known in BCa. We first discovered that
miR-515-3p inhibited cell metastasis of BCa, which could be rescued by
circSTX6. The discovery of
circSTX6/
miR-515-3p/
SUZ12 axis represents a promising step for therapeutic intervention against tumors.
Apart from functioning as miRNA sponge, growing evidence indicated that circRNAs could interact with RBPs to participate in the regulation of tumor-related genes. The patterns of circRNA-protein interactions include as follows: (i) protein scaffold, in which circRNAs bring different proteins into proximity [
58]; (ii) protein recruiter, in which specific circRNAs may recruit proteins to specific subcellular compartments [
59]; (iii) protein decoy, in which circRNAs compete for binding to specific RBPs with other molecules sharing RBP binding domains [
60]; (IV) protein function enhancer, in which individual circRNAs bind to and promote the functions of its RBPs [
61]. For instance,
circNDUFB2 functions as a scaffold to enhance the interaction between TRIM25 and IGF2BPs, which is enhanced by m
6A modifications of
circNDUFB2 [
35]. Another study identified that
circRHOT1 induces the expression of NR2F6 by recruiting TIP60 to the promoter region of
NR2F6, thereby initiating
NR2F6 transcription [
62]. Yang et al. found that
circPTK2 functions as a protein decoy by blocking the phosphorylation sites of the vimentin protein to protect it from phosphorylation, leading to upregulation of vimentin [
63]. In addition, m
6A-modified
circNSUN2 acts as an enhancer of protein function by forming a ternary complex with IGF2BP2 protein and the
HMGA2 mRNA, thus promoting the stability of
HMGA2 mRNA [
61]. In this study, we analyzed the
circSTX6 interacting protein through the online database and RNA pulldown experiments. Following a series of experiments, we further confirmed that circSTX6 could bind the RRM1-2 domain of PABPC1 protein to increase its interaction with SUZ12, resulting in promotion of SUZ12 mRNA stability. Significantly, our study demonstrates that
circSTX6 drives BCa metastasis and CDDP resistance by increasing SUZ12 mRNA expression and stability in a ceRNA- and RBP-dependent manner, which extends our knowledge of circRNAs in regulation of mRNA through dual-faceted regulation pathway.
Increasing evidence demonstrated that RNA drugs, including mRNA, shRNA, siRNA and ASO, have been approved to enter the clinical trial stage or clinical practice [
64,
65]. Our study found that shRNAs targeting
circSTX6 suppressed BCa metastasis and CDDP chemoresistance, which could serve as a feasible treatment strategy for BCa. However, safety and targetability of these agents remains a major challenge. Previous study has identified small molecular compounds 5 and 16 specifically targeting the lncRNA MALAT1 [
66], suggesting that ncRNAs can be targets for small-molecule drugs. Thus,
circSTX6 may be a potential target for developing small-molecule drugs to overcome metastasis and cisplatin resistance, which could shed light on the individual management of BCa.
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