Background
Bladder cancer (BC) ranked the 9th most common cancer in the world, with a significant morbidity and mortality [
1]. According to the Global Cancer Statistics, about 79,030 new cases of bladder cancer are diagnosed annually in the United States, and an estimated 16,870 patients will die of this disease [
2]. While the most of first diagnosed bladder cancers present as noninvasive early tumors, up to one-third of non-muscle invasive bladder cancer (NMIBC) will progress to muscle invasive bladder cancer (MIBC) and metastasize to other organs over time [
3], which highlights the urgent need for novel biomarkers and pathways to more accurately predict bladder cancer recurrence and cancer treatment.
The existence of circRNAs was first observed in eukaryotic cells nearly 40 years ago by using an electron microscope [
4]. Initially, circRNA was occasionally reported and misinterpreted as a by-product of aberrant RNA splicing or splicing errors [
5,
6]. With the advent of high-throughput sequencing, thousands of circRNAs have been successfully identified in different cell lines and species [
7]. However, little is known about their potential function and biogenesis process. Recently, circRNAs have been verified to be associated with several diseases such as brain dis-function or neurodegenerative diseases like Alzheimer’s disease and several cancers. Unlike linear RNAs, circRNAs have the prominent feature of non-canonical splicing with no free 3′ and 5′ end, which enables them to be resistant to RNA exonucleases [
8,
9]. These observations suggest that circRNA may be a novel potential biomarker and therapeutic target for cancer. However, the elucidation of deregulated circRNAs and the identification of their functions remain an ongoing process in cancer investigation.
The dysregulation and function of microRNAs (miRNAs) have been extensively studied in almost every biological process. However, the expression profile and function of newly identified circRNAs in specific biological activities still need further investigation. Pandolfi et al., reported that RNAs can co-regulate each other as ceRNAs through competitively shared miRNAs [
10]. Transcripts such as mRNAs, lncRNAs and pseudogenes can function as natural miRNA sponges by competitive binding with miRNA response elements (MREs) to inhibit their expression and function [
11]. lncRNAs acting as ceRNAs have been confirmed by several studies, while circRNAs containing multiple MREs can also serve as highly effective miRNA sponges that regulate gene expression at the transcriptional or post-transcriptional level [
12,
13]. The expression of circRNA is strictly regulated in different environments and the study of circRNA is still in its beginning. However, the role of circRNA in bladder cancer has not been fully elucidated.
In our study, we analyzed the expression profile of circRNAs in BC tissues and determined that circRNA cTFRC was significantly up-regulated in BC tissues and closely related to the prognosis of BC patients. We found that cTFRC may function as the sponge of miR-107 to up-regulate the expression of TFRC (transferrin receptor) and consequently promote BC progression. Therefore, cTFRC can serve as a biomarker for prognosis predication and as a potential therapeutic target for BC patients.
Methods
Human tissues and cell lines
Primary human BC and paired adjacent normal bladder tissues were obtained from patients during operation. With the guidance of a skillful pathologist, we collected normal bladder urothelium samples with a distance of ≥3 cm from the edge of the bladder cancer tissue. After surgical resection, all specimens were immediately frozen in liquid nitrogen. All human studies were reviewed and approved by the IRB of Institute of Biophysics, Chinese Academy of Sciences, and written informed consent was provided according to the World Medical Association Declaration of Helsinki. Clinicopathological classification and staging were determined according to the American Joint Committee on Cancer Classification Criteria. RNA expression profiles and matching clinical data on 433 bladder cancer patients were downloaded from the TCGA data portal (
https://tcga-data.nci.nih.gov/). Bladder cancer cell lines EJ, T24, 5637, UMUC3, BIU87, J82, SW780 and bladder normal epithelial cell HCV29 were cultured in RPMI 1640 medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U/mL penicillin (Gibco) and 100 μg/mL streptomycin (Gibco). All the cells were incubated at 37 °C in a humidified atmosphere containing 5% CO
2.
Expression profile analysis of circRNAs
The circRNAs chip (Arraystar Human circRNAs chip; ArrayStar, Rockville, MD, USA), containing 5396 probes specific for human circRNAs splicing sites, was used. After hybridization and washing with samples, three pairs of BC samples (tumor tissues and matched nontumor tissues) were analyzed on the circRNAs chips. Exogenous RNAs developed by the External RNA Controls Consortium were used as controls.
Real-time qRT-PCR analysis
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration was measured by Nonodrop, and each paired sample was adjusted to the same concentration. Real-time qPCR was performed as described [
14]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were used as the internal control. Primers sequences for the detected genes were listed in Additional file
1: Table S1.
Plasmids construction and stable tansfection
Short hairpin RNA (shRNA) of cTFRC were synthesized by GenePharma (Shanghai, China), shcTFRC targeting to the junction region of the cTFRC sequence. The shRNA plasmid of TFRC was purchased from Santa Cruz (Dallas, USA). EJ and T24 cells were transfected with cTFRC and TFRC shRNAs plasmids using Lipofecamine 2000 (Invitrogen, Carlsbad, CA, USA). The sequences of the effective shRNAs were provided in Additional file
1: Table S2. The full-length cTFRC cDNA was cloned into pCDH-CMV-MCS-EF1-GFP + Puro (Geneseed Biotech, Guangzhou, China) to obtain the pCDH-cTFRC overexpression of cTFRC. Production of lentiviral particles and transduction of BC cells was performed as described [
14].
Biotin-labeled pull-down assay
Biotinylated cTFRC and miR-107 (GenePharma, Shanghai, China) pull-down assay with target mRNAs was performed as described earlier [
15]. In brief, 1 × 10
7 bladder cancer cells were harvested, lysed, and sonicated. The probe was incubated with probes-M280 streptavidin dynabeads (Invitrogen) at 25 °C for 2 h to generate probe-coated beads. The cell lysates were incubated with the probe-coated beads mixture at 4 °C overnight. After washing with the wash buffer, the RNA complexes bound to the beads were eluted and extracted with Trizol Reagent (Invitrogen) for the analysis.
Western blots
Cell lysates were prepared with RIPA buffer (Thermo Scientific). Immunoreactive bands were detected by using the Immobilon ECL substrate kit (Millipore, Merck KGaA, Germany). Antibodies used included primary antibodies against TFRC (Cat. No: ab218544, 1:1000 dilution, Abcam, USA), E-cadherin (Cat. No: ab1416, 1:1000 dilution, Abcam, USA) and β-actin (Cat. No: ab8226 1:1000 dilution, Abcam,); HRP-conjugated secondary goat anti-mouse (Cat. No: SA00001–1) or goat anti-rabbit (Cat. No: SA00001–2) antibodies (1:4000 dilution, Proteintech, USA).
Cell invasion assay
The invasion assays were performed in 24-well FluoroBlok cell culture inserts (BD Biosciences) with 8-μm pore-size PET membrane. The insert was coated with 100 μL of 1 μg/μL Matrigel matrix (BD Biosciences) at 4 °C overnight. Following starvation for 6 h in serum-free RPMI 1640, cells were harvested from one subconfluent 10-cm dish by cell dissociation buffer (Life Technologies), spun at 500×g for 3 min, and resuspended in RPMI 1640. Cells (4 × 104) in 500 μL of RPMI 1640 were seeded onto the insert and 750 μL of RPMI 1640 with 10% (vol/vol) FBS was added into the lower chamber of the transwells. After incubation for 18 h at 37 °C, the medium inside the insert was removed and the insert was then placed in a new 24-well plate. The invaded cells at the reverse side of the insert were labeled with a fluorescent dye Calcein AM (4 μM in Dulbecco’s PBS) (BD Biosciences) for 1 h at 37 °C. The fluorescence was measured with 494 nm/517 nm (excitation/emission wavelength) by a SpectraMax M5 microplate reader (Molecular Devices).
[3H] thymidine incorporation
Cells were planted in 96-well plates and grown for 24 h after they were serum starved for 48 h. They were treated with 15d-PGJ2 for 48 h and pulsed with 5 μCi of [3H] thymidine for 4 h. We counted the radioactivity in Beckman L5 counter after washing the cells and stopping the reaction with 5% trichloroacetic acid and solubilizing the cells in 0.5% of 0.25 N sodium hydroxide. Each experiment was done in quadruplicates and repeated at least three times.
Prediction of miRNA targets
CircRNA-miRNA interaction was predicted with miRNA target prediction software (Arraystar’s home-made) based on TargetScan and miRanda. TargetScan (
http://www.targetscan.org) or miRBase (
http://www.mirbase.org) were used to identify the miRNA targeting sites in TFRC 3’-UTR.
Luciferase reporter assay
For luciferase reporter assay, pmirGLO Dual-luciferase vectors (GenePharma, Shanghai, China) were used to construct dual luciferase reporter plasmids. EJ or T24 cells were co-transfected with corresponding plasmids and microRNA, luciferase activity was assessed using the dual-luciferase reporter kit (Promega, Madison, WI, USA). The relative firefly luciferase activity was normalized to Renilla luciferase activity.
Mice model
All animal studies were permitted by the Institutional Animal Care and Use Committee of the Institute of Biophysics, Chinese Academy of Sciences and were conducted in compliance with its recommendations. The stably knockdown of cTFRC EJ cells (2 × 106) or T24 cells (2 × 106) were subcutaneously injected into the back of 4-week-old male BALB/c mice. The tumor volume was monitored every 5 days. After 30 days, the mice were sacrificed and tumor tissues were excised and subjected to pathologic examination.
Fluorescence in situ hybridization
Hybridization was performed overnight with cTFRC and miR-107 probes. Specimens were analyzed on a Nikon inverted fluorescence microscope. The cTFRC and miR-107 probe for fluorescence in situ hybridization (FISH) were listed in Additional file
1: Table S3.
Statistical analysis
The Student t test was performed to analyze whether two experimental groups have significant difference using P < 0.05 as the significant criteria. Survival analysis was performed by Kaplan-Meier curves and log-rank test for significance in GraphPad Prism 5.
Discussion
Numerous studies have shown that the expression profiles of non-coding RNAs (including lncRNAs and miRNAs) are abnormal in many types of cancers, and many of them focus on their epigenetic regulation in the development of cancer [
22]. Recent studies have reported that many miRNAs and several lncRNAs may play a regulatory role in the development of BC [
23,
24]. However, it is unclear whether circRNA plays a role in BC. In the past few years, the presence of circRNA has occasionally been recognized with covalent linkages in animal cells. Previously it was thought to be rare and even considered as transcriptional noise and artifacts [
25]. However, circRNAs have recently been identified to be abundant stable ncRNAs by high-throughput sequencing and bioinformatics analysis [
26]. It has been reported that circRNA is dysregulated in different cancer types such as colorectal cancer [
27], liver cancer [
19], esophageal squamous cell carcinoma [
28], basal cell carcinoma [
29] and laryngeal cancer [
30]. These differentially expressed circRNAs may have some potential functions in the regulation of gene expression and have been widely accepted. CircRNA microarray results provide useful information for screening differentially expressed circRNAs and help us to select candidate circRNAs for further study.
Here, we identified a novel circular RNA through circRNA microarray analysis termed cTFRC that was significantly upregulated in human BC and correlated with clinical stage. Functionally, we found that down-regulation of cTFRC could inhibit cell invasion and proliferation, reduce EMT as well as facilitate tumor growth in vivo, whereas upregulation of cTFRC exerted an inductive role in EMT. Mechanistically, cTFRC could function as a ceRNA through harboring miR-107 to abolish the suppressive effect on the target gene TFRC in bladder cancer progression. Thus, our data suggest that cTFRC could play an important role in the pathogenesis and development of BC.
The role of circRNA in carcinogenesis and cancer progression has not been elucidated clearly. CircRNA may regulate the expression of oncogene or tumor suppressor gene through different targets, depending on the cancer or even at different stages. Together with miRNA and their target gene, the circRNA-miRNA-mRNA axis may serve as a wide network of gene expression regulators, and its deregulation may lead to disease progression, including cancer development. For example, circRNA SRY was reported to be a tumor-associated molecule in colorectal cancer and ovarian cancer by absorbing miR-138 [
7]. It has been reported that circHIPK3 promotes proliferation of human liver cancer HuH-7 cells, human colon cancer HCT-116 cells, and human cervical cancer HeLa cells via sponging multiple miRNAs [
31]. This assumption may lead to some interesting future work to elucidate the regulatory networks of non-coding RNAs and coding genes in cancer biology.
Although we have made encouraging progress in understanding the molecular mechanisms of BC, the prognosis of patients with advanced bladder cancer remains unfavorable. Therefore, it is of great significance to reveal the underlying mechanism of bladder cancer metastasis. Specifically, EMT is a powerful paradigm for studying the genetic progression of advanced stage solid tumors [
32]. In addition, as most human malignancies originate from epithelial tissues, the investigation on EMT is not only beneficial to bladder cancer, but also to other solid tumors [
33]. In the present study, we explored cTFRC on the EMT process of bladder cancer cell by regulating TFRC expression. Underexpression of cTFRC induced EMT process marker E-cadherin expression and suppression TFRC expression. Besides, overexpression of cTFRC resulted in morphological alteration and EJ-oecTFRC or T24-oecTFRC cells displayed elongated mesenchymal-like characteristics, indicating that these cells were undergoing epithelial to mesenchymal transition. Changes in morphology of EJ or T24 cells might occur after cTFRC regulates TFRC expression.
TFRC plays a pivotal role in iron cellular uptake, and cellular iron deficiency inhibits cell growth and leads to cell death [
34]. In malignant tissues, TFRC expression is more than in their normal tissues, as cancer cells require large amounts of iron to maintain their high rate of cell proliferation [
35]. Therefore, TFRCs are attractive targets for immunotherapy and cytotoxic delivery agents because of their increased expression on malignant cells as compared to normal cells. Recently, many reports found that TFRC is involved in tumor progression [
36]. However, TFRC has not been reported on bladder cancer progression. In this study, we found that TFRC is overexpressed in bladder cancer and correlated with poor prognosis of BC patient. More interesting is that cTFRC expression is correlated with TFRC both in tumor cell lines and tumor tissues. Also, we found that cTFRC was overexpressed in bladder cancer and cTFRC up-regulated TFRC expression in bladder cancer as a “miRNA sponge”. Thus, this may explain the high expression of TFRC in BC patients, but little is known about cTFRC highly expressed in bladder cancer. Therefore, the regulation of cTFRC expression in BC also requires further exploration.