Background
Bladder cancer (BC) is the number one malignancy of urinary system with an estimated over 79,030 deaths predicted in 2017 in the United State [
1].The high rate of recurrence and distant metastasis of BC created a huge economic burden in EU [
2]. New technology like the blue-light cystoscopy has been proved to improve the detection of BC, especially flat lesions [
3]. However, the researches on early diagnostic assessment and specific markers for BC are still deficient [
4]. The guideline provides recommended treatment based on the grade and stage of BC [
5,
6], ranging from radical cystectomy to systemic chemotherapy. Nevertheless, the overall therapeutic effects of BC are limited and the five-year survival rate keeps at a low level [
7,
8]. Thus, further exploration of genetic regulatory networks involved in BC progression and development of precise strategies are worthy and important.
Circular RNAs (circRNAs), a new member of noncoding RNAs (ncRNAs), have attracted great attentions for their closed continuous loop structure and potential value in clinical work [
9,
10]. CircRNAs were found in cells in the 1970s by electron microscope [
11,
12]. The development of the high-throughout sequencing and computational approaches have identified more than 30,000 circRNAs and proved that they are endogenous, abundant and conserved in mammalian cells [
13‐
15]. Importantly, studies have demonstrated that circRNAs are closely related to neurological disorders, atherosclerotic vascular disease risk, carcinomas and so on [
16‐
18]. Some circRNAs contain miRNA response elements (MREs) and function as competing endogenous RNAs (ceRNAs) to interact with miRNAs and regulate the expression of target mRNAs. The studies of CiRS-7 provided the solid evidence for this notion [
16,
19]. CiRS-7 has more than 70 miR-7 binding sites and thus acts as effective miR-7 suppressor to regulate the expression of miR-7 target mRNAs. Recently, circBIRC6 has been found to directly interact with miR-34a and miR-145 to modulate target genes that maintain pluripotency [
20], and it is reported that CircMTO1 suppresses human hepatocellular carcinoma progression by acting as the sponge of oncogenic miR-9 to promote p21 expression [
21]. In general, the circRNA-miRNA-mRNA axis may function as an extensive regulatory network in progression of some diseases.
In our previous study, we found 6154 distinct circRNAs from human BC and normal bladder tissues by performing RNA-seq, and identified circHIPK3 as a tumor suppressor in BC [
22]. CircHIPK3 inhibits migration, invasion, and angiogenesis of BC cells via acting as “miRNA sponge” for miR-558. In this research, we mainly focus on the impacts of circRNAs on BC cell proliferation and characterize a circRNA derived from PSMD1 gene (bladder cancer related circRNA-3, BCRC-3). BCRC-3 is significantly down-regulated in BC tissues and effectively inhibits the proliferation of BC cells. Importantly, our study reveals that BCRC-3 could bind to oncogenic miR-182-5p to promote p27 expression and therefore inhibit BC progression. Collectively, BCRC-3 may serve as a novel promising target for BC treatment.
Methods
Patient tissue specimens and cell lines
A total of 47 BC tissues and their adjacent normal bladder tissues were obtained from patients who underwent radical cystectomy for urothelial carcinomas of bladder at the Department of Urology of Union Hospital affiliated of Tongji Medical College between 2015 and 2017. We have received the approval from the Institutional Review Board of Tongji Medical College of Huazhong University of Science and Technology before we collected the samples. All specimens were classified according to the 2004 World Health Organization Consensus Classification and Staging System for bladder neoplasms. Clinicopathological characteristics of patients are shown in Additional file
1: Table S1. The human BC cell line EJ and normal human urothelial cells SV-HUC-1 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The human BC cell line T24 T was provided by Dr. Dan Theodorescu (Departments of Urology, University of Virginia, Charlottesville, VA) as described in our previous studies [
23]. All the cell lines were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Australia origin) and 1% penicillin/streptomycin (Gibco) in the recommended media at 37 °C supplied with 5% CO
2.
Total RNA was isolated from tissues and cell lines with RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. RNase R treatment was processed at 37 °C with 3 U/mg of RNase R (Epicenter, WI, USA) for 15 min. Complementary DNA was synthesized using random primers and the reverse transcription kit PrimeScript RT Master Mix (Takara, Dalian, China) or commercial miRNA reverse transcription PCR kit (RiboBio, Guangzhou, China). Genomic DNA (gDNA) was isolated with QIAamp DNA Mini Kit (QIAGEN, Germany). Quantitative real-time PCR (qRT-PCR) analysis was carried out using the SYBR Premix Ex TaqTM kit (Takara). The differences of circRNA and miRNA were normalized to the levels of GAPDH or U6. All data were analyzed via the StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). Bulge-Loop miRNAs qPCR primers were obtained from RiboBio. The details of primers are listed in Additional file
1: Table S2.
Plasmids construction and stable transfection
The human BCRC-3 and p27 3’UTR cDNA was synthesized by TSINGKE (Wuhan, China). BCRC-3 was cloned into pCD-ciR vector (Geenseed Biotech Co, Guangzhou, China) which contained a front circular frame and a back circular frame. P27 3’UTR was cloned into pMIR-REPORT vector. The plasmids of p27 3’UTR T1, T2, T3 and mutant luciferase reporters were synthesized using Trelief™ SoSoo Cloning Kit (TSINGKE, Beijing, China). The p27 promoter luciferase reporter vector was constructed and used as our previous study described [
24]. MiR-182-5p mimics and its control were purchased from RiboBio (Guangzhou, China). SiRNA aimed at BCRC-3 was synthesized by Gene-Pharma (Shanghai, China). ShRNAs targeting p27 were designed and synthesized by Genechem (Shanghai, China). Lipofectamine 2000 (Life Technologies) was used for plasmid transfection following the manufacturer’s instructions. The cells transfected BCRC-3 were screened with G418 (Life Technologies) for 4–6 weeks.
RNA –FISH
Cy3-labeled BCRC-3 and Dig-labeled locked nucleic acid miR-182-5p probes were purchased from RiboBio (Guangzhou, China). The images were obtained using Fluorescent in Situ Hybridization kit (RiboBio) following the manufacturer’s instructions. All data were analyzed via Nikon A1Si Laser Scanning Confocal Microscope (Nikon Instruments Inc., Japan).
Flow cytometry assay for the cell cycle
EJ and T24 T cells transfected with the plasmids or treated with MJ were harvested and stained with propidium iodide buffer (BD Pharmingen) for cell cycle analysis. The results were analyzed by the ModFit LT software.
EJ and T24 T cells transfected with the plasmids were cultured in 6-wells plates at density of 800–1000 cells per well. Plates were incubated at 37 °C in 5% CO2 for 2–3 weeks, and the colonies with more than 50 cells were scored. The cell colonies were immobilized with 4% paraformaldehyde and dyed by coomassie brilliant blue.
5-Ethynyl-20-deoxyuridine (EdU) assay
Cell-Light EdU DNA Cell Proliferation Kit (C10310–1, RiboBio Guangzhou, China) was used to assess cell proliferation viability following the manufacturer’s protocol. All images were obtained with an Olympus FSX100 microscope (Olympus, Tokyo, Japan). The ratio of EdU-stained cells to Hoechst-stained cells was calculated to evaluate the cell proliferation as described [
25].
Tumor xenografts
We chose 4-week-old female BALB/c nude mice for tumor xenografts experiments. T24 T cells stably transfected with BCRC-3 plasmids or control vector were subcutaneously injected into the upper back of the nude mice (3 × 106, 200 μl). Mice were sacrificed and detected for tumor weight, gene expression after one month. All procedures were approved by the Animal Care Committee of Tongji Medical College.
Western blotting analysis
The proteins were extracted in RIPA lysis buffer (Thermo Scientific) and determined using BCA Protein assay kit (Beyotime). After separated by electrophoresis and transferred onto PVDF membranes, total proteins were incubated with primary antibodies overnight. The membranes were blocked for 1 h in the specific HRP-conjugated secondary antibodies at room temperature. All images were obtained by using BioSpectrum 600 Imaging System (UVP, CA, USA). Antibodies against CDK2 (Cat No: 10122–1-AP), CDK4 (Cat No: 11026–1-AP), CDK6 (Cat. No: 14052–1-AP), cyclin D1 (Cat. No: 60186–1-Ig), Cyclin E (Cat. No: 11554–1-AP), p21 (Cat. No: 10355–1-AP), p27 (Cat. No: 25614–1-AP), β-actin (Cat. No: 60008–1-Ig), HRP-conjugated secondary goat anti-mouse (Cat. No: SA00001–1) and goat anti-rabbit (Cat. No: SA00001–2) were purchased from Proteintech Group (Chicago, USA).
Pull-down assay with biotinylated BCRC-3 probe
Biotinylated-BCRC-3 probe was synthesized by RiboBio (Guangzhou, China). The sequence of the probe was just complemented to the back-spliced junction of BCRC-3 (listed in Additional file
1: Table S2). Pull-down assay was carried out as described in our previous study [
22]. The RNA complexes combining on the beads were finally extracted with RNeasy Mini Kit (QIAGEN, China) for further research.
Pull-down assay with biotinylated miRNA
Biotinylated miRNA mimics or their mutants were synthesized by RiboBio (Guangzhou, China). The sequences are listed in Additional file
2: Figure S4b. The pull-down assay with biotinylated miRNA was performed as described in our previous study [
22]. The bound RNAs were purified using RNeasy Mini Kit (QIAGEN) for further analysis.
Immunohistochemistry analysis
The primary antibody used to detect p27 was purchased from Proteintech Group (Chicago, USA). The immunostaining images were captured using Olympus FSX100 microscope (Olympus, Japan). Protein expression levels were analyzed by calculating the integrated optical density as described [
26].
Luciferase reporter assays
The p27 3’UTR or promoter reporters were transiently transfected along with Renilla control plasmid, and the BC cells were co-tranfected with BCRC-3, miR-182-5p mimics, or co-treated with MJ, respectively. The luciferase activities were measured following dual luciferase reporter assay detection kit (Promega, WI, USA) as described [
27] after 24 h.
Statistical analysis
All data were indicated as means ± standard error of the mean (SEM) processed by GraphPad Prism 5.0 (La Jolla, USA). Student’s t-test or chi-square (P < 0.05) was used to evaluate the Group difference.
Discussion
During the past years, hundreds of circRNAs have been reported to function as important drivers of tumorigenesis or tumor suppressors in distinct human cancers [
29]. One important function pattern for circRNAs is acting as inhibitors of miRNAs by direct binding. CircRNAs which were regarded as miRNA sponge have some characteristics in common. Firstly, they derive from one or more exons of known protein-coding genes through back-splicing [
30]. Second, subcellular location of these circRNAs in cell lines is predominantly in the cytoplasm, which occupies the same space of miRNA [
31]. Finally, circRNAs with more predicting putative miRNA binding sites are likey to play the role of ceRNA for miRNA [
21]. Our High-Throughout Sequencing had confirmed 524 down-regulated circRNAs and 47 up-regulated circRNAs in BC tissues [
22]. Subsequently, we focused on the down-regulated circRNAs first, and in this study, we identified BCRC-3 which consists of nine exons (1,002 bp) from the PSMD1 gene, mainly locating in the cytoplasm. Our miRNA-targeting analysis showed that BCRC-3 harbors 3 targeting sites for miR-182-5p, suggesting that BCRC-3 may act as a miRNA sponge. RNA FISH demonstrated that BCRC-3 and miR-182-5p are co-localized in cells. Therefore, our results provided further evidence to support the notion that circRNAs can regulate gene expression via acting as “miRNA sponge”. It is suggested that exogenous expression of lowly expressed circRNAs can be performed by gene therapy where DNA cassettes designed for circRNA expression are delivered [
32]. On the other hand, it is worthy to explore the roles of the up-regulated circRNAs to find novel therapeutic targets for the clinical application of BC.
To date only a few circRNAs with multiple binding sites for a single miRNA have been discovered and many circRNAs may have other roles in regulating cellular function. Like other noncoding RNAs, some circRNAs could function as protein decoys. With regard to this aspect, circFoxo3 harbors binding sites for p21 and CDK2. As a result, this ternary complex blocked cell cycle progression [
33].Another interesting example is circPABPN1, which is derived from the PABPN1 gene [
34]. The competitive binding of circPABPN1 to HuR prevents HuR binding to PABPN1 mRNA and reduces its translation. Unlike most circRNAs, circular intronic RNAs (ciRNA) and EIciRNAs are usually retained in the nucleus, where they may regulate transcription or alternative splicing [
35]. Apart from the functions mentioned above, some endogenous circRNAs with an IRES or open reading frames have potential abilities to translate proteins with the help of modifications [
36,
37], which challenges the conventional concept of non-coding RNAs. Therefore, apart from miRNA sponge, other functions of BCRC-3 still need to be elucidated in BC cells.
P27 (CDKN1B) is firstly identified as a negative regulator that halts cycle progression at G1/S phase. Abundant interacting proteins of p27 have been identified in recent years, indicating new roles for p27 in several CDK-unrelated processes [
38,
39]. It is verified that the function of p27 protein is mainly regulated by post-translational modifications, especially phosphorylation of particular amino acid [
40,
41], which alter the cellular localization and the degradation. However, the expression of p27 protein could also be regulated by transcriptional and posttranscriptional mechanisms. Accordingly, human tumors with an abnormal p27 metabolism/localization show poor prognosis and decreasing median survival time. Our recent studies demonstrated that ectopic expression of miR-182-5p blocked p27 3’UTR activity, whereas mutation of the binding sites at p27 3’UTR effectively reversed this inhibition [
42]. Our data presented here indicated that BCRC-3 could interact with miR-182-5p to promote the expression of p27. Hence, we provided further evidence for the posttranscriptional regulation of p27 by circular RNA in cancer cells.
Among the naturally occurring jasmonates and their synthetic derivatives, Methyl jasmonate (MJ) showed the highest activity in accordance with cytotoxicity and induction of apoptosis in human cancer cells [
43‐
45]. Besides, increasing evidence indicated that MJ could suppress the proliferation of urological malignancy [
28,
46] depending on cellular mRNA transcription and protein translation [
47,
48]. We have reported that MJ possessed high selectivity anticancer function toward BC cells by inducing apoptosis [
28]. In the present study, we explored the effects of MJ on BC cell cycle progression and demonstrated that MJ could promote p27 expression through BCRC-3/miRNA-182-5p/p27 axis. Nevertheless, the mechanism underlying MJ-induced BCRC-3 expression still needs to be investigated in future studies.
Conclusions
In summary, we firstly demonstrate that BCRC-3 is down-regulated in BC tissues and cell lines for the first time. BCRC-3 is capable of functioning as ceRNA for miR-182-5p to regulate the expression of p27. Moreover, our results show that cytotoxic MJ boosts the p27 protein via increasing the BCRC-3 expression, thus inhibiting the proliferation of BC cells. Our results not only explain the potential mechanisms related to circRNA in regulation BC cell proliferation, but they also make circRNA as a promising therapeutic target for BC treatment.