Introduction
Bladder cancer is a common type of cancer involving tumors of the urinary system, has the highest published incidence involving malignant urinary system tumors, and poses a serious threat to human health. According to data released by the US Department of Health, an estimated 76,960 patients were diagnosed with bladder cancer and 16,390 died of complications in 2016 [
1]. The most common histopathological type of bladder cancer is transitional cell carcinoma (TCC), which accounts for more than 90% of bladder cancers, followed by squamous cell carcinoma, adenocarcinoma, and undifferentiated carcinoma [
2]. Bladder cancer is characterized by multifocality and relapse. Most patients have non-muscle invasive bladder cancer at initial diagnosis. After active surgery and bladder perfusion therapy, relapse, infiltration and drug resistance can occur, which can threaten human health and socio-economic development [
3]. The lack of specificity and sensitivity of technologies in the early diagnosis of bladder cancer, as well as the high rates of postoperative recurrence and malignant transformation following surgery, such as bladder tumor resections, are the major problems in bladder cancer diagnosis and treatment. Therefore, identification of new, highly sensitive, specific and cost-effective bladder cancer markers is needed to improve the early diagnosis rate of bladder tumors, which will have important clinical significance for ultimately improving the prognoses of patients.
LncRNAs are non-coding RNAs that are more than 200 nucleotides in length and that affect regulatory gene expression. LncRNAs lack a complete open reading frame and do not have a protein coding function. LncRNAs were first discovered by Okazaki et al. [
4]. By using mouse DNA transcripts. Up to 4–9% of the sequences in the mammalian genome sequence can produce lncRNAs. LncRNAs were originally thought to be the “dark matter” or “noise” of genomic transcription and to have no biological function [
5]. Later studies demonstrated that lncRNAs are involved in many important cellular functions, such as X chromosome silencing, genomic imprinting, chromatin modification, and transcriptional activation or inhibition, and can promote changes in molecular function in related signaling pathways, as well as alter cell life activities [
6‐
8]. LncRNA plays a key role in the initiation, development and metastasis of bladder cancer. At present,
UCN-1,
PVT-1,
MALAT1,
SPRY4-IT1,
PANDAR,
H19 and other lncRNAs closely related to bladder cancer have been identified. These lncRNAs affect important biological roles, such as proliferation, apoptosis, migration and invasion of bladder cancer, and also participate in disease progression and outcomes by regulating epigenetic modifications and key cell signaling transduction pathways. In this study, gene chip screening technology was used to discover lncRNAs related to the occurrence and development of bladder cancer and to identify their functions and regulation mechanisms to promote a new understanding of the pathogenesis of bladder cancer and to guide clinical treatment.
Methods
Microarray profiling
TRIzol Reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA which was then purified by a RNeasy Mini Kit (Qiagen, Valencia, CA). Differentially expressed lncRNAs in BC and normal adjacent tissues were screened by the LncRNA microarray expression profiling based on the criteria of log2 (fold change) > 1.5 and adjusted P < 0.01. Manufacturer’s standard protocols were strictly followed. Briefly, cDNA was synthesized, labeled and purified. lncRNA microarray chips was hybridized by Cyanine-3-CTP labeled cRNA. Then after washing, samples were analyzed on the lncRNAs microarray. The differentially expressed genes were calculated and clustered by R program.
Tissue samples
Resected BC and normal adjacent tissues were collected from The Third Affiliated Hospital of Soochow University from Jan 2013 to Jan 2016. There were 13 BC samples and 8 normal adjacent tissue samples. All tissues were directly stored in liquid nitrogen at − 80 °C. Informed consent was obtained from each participant. The use of human clinical tissues was approved by the Institutional Human Experiment and Ethics Committee of The Third Affiliated Hospital of Soochow University. The Declaration of Helsinki was strictly followed during experiments.
Cell line culture
Cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) including BC cell lines (5637, UMUC3 and T24), human bladder epithelium immortalized cells (SV-HUC-1) and human embryonic kidney cell line (HEK-293). Cells were maintained in modified RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) including 100 μg/L penicillin and 100 μg/L streptomycin. All cell lines were grown with 5% CO2 at 37 °C.
Real-time quantitative polymerase chain reaction (RT-qPCR)
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolated total RNA from tissues and cells according to the manufacturer’s instructions. Moloney Leukemia Virus Reverse Transcriptase Kit (Promega, Madison, WI, USA) was then performed to reverse transcribe total RNA(1 μg) to cDNA. Target primers were amplified by SYBR Green Mix (Promega). Sequences of the primers are listed in Table
1. All primers were synthesized by Shanghai Tingzhou Biological Engineering Co., Ltd. The
miR-590 level was performed using TaqMan MicroRNA Assays Kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturers’ instructions. All results were calculated and expressed as 2
-ΔΔCt. GAPDH was used as endogenous control for
LINC00612 and
PHF14 and U6 for
miR-590. Triplicate is required for each experiment.
Table 1
Primer used in this study
LINC00612 forward | 5′-GGCAGAGCCATGTGTTGGATA-3′ |
LINC00612 reverse | 5′-GTGCTCCCTAATGGCTCACA-3′ |
PHF14 forward | 5′-GCAACTTGCAAGGGAACTGG-3’ |
PHF14 reverse | 5′-AAGAGGTTTCCGGGATTGCC-3’ |
GAPDH forward | 5′-TGAACGGGAAGCTCACTGG-3’ |
GAPDH reverse | 5′-TCCACCACCCTGTTGCTGTA-3’ |
U6 forward | 5′-CTCGCTTCGGCAGCACA-3’ |
U6 reverse | 5′-AACGCTTCACGAATTTGCGT-3’ |
RNA isolation of nuclear and cytoplasmic fractions
The Nuclear/Cytoplasmic Isolation Kit (Biovision) was applied to isolate and collect cytosolic and nuclear fractions. RNA levels of LINC00612, RNU6–1(nuclear control transcript) and GAPDH (cytoplasmic control transcript) were analyzed by RT-qPCR.
In situ hybridization (ISH)
Cells were seeded onto poly-L-lysine-treated glass slides for 24 h after trypsinization harvest and then fixed in methanol at − 20 °C for 5 min. The ISH assays were performed as previously described [
9]. A locked nucleic acid probe with complementarity to a section of
LINC00612 (5′- TATCGAACTTTCTAGATCGGTGCAC-3′ custom LNA detection probe, Exiqon) was labeled with digoxigenin antibody (Roche, 11,093,274, 1:1000) and synthesized. The intensity and the extent of staining were evaluated by 2 pathologists who were blinded to the experiment.
Fluorescence in situ hybridization (FISH)
Five thousand six hundred thirty-seven and T24 cells were fixed in 4% PFA for 15 min. Then, 0.5% TritonX-100 was used to permeabilize the cells for 15 min at 4 °C. Digoxigenin (DIG)-labeled LINC00612 probe or control probe mix were performed to incubate cells for 4 h at 55 °C. After 2 × saline-sodium citrate briefly washing for 5 min (5–6 times), signal was detected by Horseradish peroxidase (HRP)-conjugated anti-DIG secondary antibodies (Jackson, West Grove, PA, USA). Olympus confocal laser scanning microscope was applied for image obtaining. DAPI was used to counterstain nuclear.
IHC
IHC staining was performed as previously described [
9]. Briefly, the tumor tissues were cut into 4-mm-thick sections, dewaxed in xylene and rehydrated in a graded series of alcohols. Antigen was retrieved by heating the tissue sections at 100 °C for 30 min in EDTA solution (1 mM, pH 9.0). Cooled tissue sections were immersed in 0.3% hydrogen peroxide solution for 15 min to block endogenous peroxidase activity, rinsed with phosphate-buffered saline (PBS) for 5 min and blocked with 3% BSA solution at room temperature for 30 min. Subsequently, the sections were incubated with mouse monoclonal antibody against human
PHF14 (1:200) at 4 °C overnight, followed by incubation with HRP-conjugated goat anti-rabbit secondary antibody. Diaminobenzene was used as the chromogen, and hematoxylin was used as the nuclear counterstain.
Lentivirus production and cell transfection
The pLVX-IRES-Puro vector for LINC00612 overexpression and lentivirus-containing short hairpin RNA (shRNA) targeting LINC00612 (top strand: Top Strand 5′-CACCGGTAGATGACAGATTAGATACCGAAGTATCTAATCTGTCATCTACC-3′; bottom strand: 5′-AAAAGGTAGATGACAGATTAGATACTTCGGTATCTAATCTGTCATCTACC-3′) were purchased from Genelily BioTech Co., Ltd., (Shanghai, China). The cells were selected by puromycin (2 μg/mL) for 2 weeks at 48 h after transfection. Cell lines with stable LINC00612 silence or overexpression was then constructed. RT-qPCR was performed to verify the transfection efficiency. The miR-590 mimic, miR-590 inhibitor, and negative control (NC) oligonucleotides were obtained from Tingzhou Biological Engineering Co., Ltd. (Shanghai, China). Abovementioned oligonucleotides and plasmids were transfected by using Lipofectamine 3000 (Invitrogen). The manufacturer’s instructions were strictly followed.
Cell counting Kit-8 (CCK8) and Colony formation assay
Cells (2 × 10
4 cells/ml) were seeded onto 96-well plates (100 μL/well) and then placed in an incubator with 5% CO
2 at 37 °C for 24 h. After the cells were cultured for 5 days, 10 μl of CCK8 solution was added to each well. The absorbance values at a wavelength of 450 nm were measured to evaluate cell viability. For colony formation assay, BC cells colon spheres were generated as previously described [
10]. Briefly, cells (500 cells/well) were seeded in to 6-well plates for 24 h. Cells were incubated for 2 weeks, then fixed in methanol and stained with 0.1% crystal violet. Quantity One software (Bio-Rad, Hercules, CA, USA) was used to count colonies. Triplicate is required for each experiment.
Transwell assay
Transwell chambers (8-μm pore size; Corning Costar, Cambridge, MA, USA) was applied to measure cell invasion ability. Instruction was strictly followed. Cells were suspended in serum-free RPMI-1640 medium, then seeded into the upper chamber. Serum (20%) was supplemented into the lower chamber that containing RPMI-1640 medium which was regarded as a chemoattractant. After 48 h incubation, the filters were fixed in methanol and stained with 0.1% crystal violet. The upper faces of the filters were gently abraded. Cells migrated across the lower faces of filters were imaged and counted under the microscope. Triplicate is required for each experiment.
Western blot analysis
Western blot analyses were performed according to standard protocols as previously described [
11].
Anti-E-cadherin, Anti-N-cadherin, Anti-vimentin and Anti-PHF14 were purchased from Sigma.
Luciferase reporter assays
The reporter vector pmirGLO-LINC00612-wt was formed by cloning LINC00612 cDNA which contains predictive binding site of miR-590 into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). The vector pmirGLO-LINC00612-Mut was inserted by the mutant LINC00612 that containing point mutations of the miR-590 seed region binding site. HEK-293FT cells were cultured and co-transfected with pmirGLO-LINC00612–3′-UTR vectors including wild-type or mutant fragments, miR-590 and miR-NC. Likewise, wild-type and mutant PHF14 3′-UTR fragments were cloned into the pmirGLO vector. The miR-590 or miR-NC was co-transfected with PHF14-wt or PHF14-Mut vector into HEK-293FT cells using Lipofectamine 3000 (Invitrogen). The Dual Luciferase Reporter Assay System (Promega) was applied at 48 h after transfection according to the manufacturer’s instructions. Triplicate is required for each experiment.
RNA immunoprecipitation
The EZMagna RIP Kit (Millipore) was applied according to the manufacturer’s protocol. Complete RNA immunoprecipitation (RIP) lysis buffer was used to lyse BC cells. Magnetic beads conjugated with Anti-Argonaute 2 (AGO2) or control anti-IgG antibody were performed in incubation of cell extract. The cell extract was incubated for 6 h at 4 °C. Then RT-qPCR analysis for purified RNA was conducted as removal of proteins of the beads had been done.
RNA pull down assay
Briefly, T24 cells were transfected with the 3’end biotinylated
miR-590 or
miR-590-mut or several candidate miRNAs for 24 h at a final concentration of 20 nmol/L. Then, the cells were incubated in the cell lysate with streptavidin-coated magnetic beads (Ambion, Life Technologies). The biotin-coupled RNA complex was pulled down and analysis of the abundance of
LINC00612 in bound fractions was then conducted by RT-qPCR. The pull-down assay was performed as previously described [
12].
Xenograft tumor model
Xenograft tumor model was performed in BALB/c-nude mice (4–5 weeks of age) which were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., China. The experimental procedures were approved by the Institutional Animal Care and Use Committee of our institution. Tumor growth was monitored every 5 days; tumor volumes were estimated by length and width. One month later, the mice were sacrificed, then tumors were excised and weighed.
After anesthetization, a left lateral flank incision was operated on the mice. The spleen was then exteriorized. About 100 μl of Hank’s balanced salt solution that containing T24-Luc-vector and T24-Luc-
siLINC00612 cells (8 × 10 [
6]) were injected into the spleen parenchyma by 25-gauge needle. The IVIS bioluminescence imaging system (Caliper Life Sciences) was performed to collect bioluminescence images after day 28. All experiments were approved by the relevant guidelines of The Third Affiliated Hospital of Soochow University.
Statistical analysis
SPSS 22.0 statistical software package and GraphPad Prism 7.0 were applied for statistical analyses.
All data are represented as mean ± standard deviation (SD). To compare two or more groups, the Student’s t-test or one-way analysis of variance (ANOVA) were performed for differences analysis. Differences were considered statistically significant when P < 0.05.
Discussion
In recent years, several lncRNAs related to the development of bladder cancer have been identified. Wang et al. [
16] reported that exogenous expression of urothelial cancer associated 1 (
UCA1) in the bladder transitional cell carcinoma cell line BLS-211 enhanced the proliferation, migration, invasion and drug resistance of bladder cancer cells. Wu et al. [
17] reported that
UCA1 can regulate the proliferation of bladder cancer cell lines through the PI3KAKT-mTOR signaling pathway. Furthermore, they also confirmed that the transcription factor CCAAT/enhancer-binding protein (C/EBP)-α can affect the expression level of
UCA1.
UCA1 inhibits the growth of bladder cancer cells by inhibiting the expression of the transcription factor C/EBP-α. In addition, a number of studies have shown that
UCA1 expression can be detected in tumors of bladder cancer patients, as well as in their urine, and exhibits tumor tissue specificity [
18‐
20].
UCA1 exhibits high sensitivity, strong specificity, and stable experimental results with respect to bladder cancer. Chen et al. [
21] used microarray analysis to screen differentially expressed lncRNAs and demonstrated that the expression level of the lncRNA
n336928 in bladder cancer tissues was significantly higher than that in paracancerous tissue and that expression of the lncRNA
n336928 was positively correlated not only with the stage and grade of bladder tumors but also with age, gender, smoking status, tumor size, and tumor number. Survival analyses revealed that the 5-year survival rate of patients in the high lncRNA
n336928 expression group was significantly lower than that in the low expression group. Liu et al. [
22] reported that expression of
SPRY4-IT1 in bladder cancer tissues and bladder cancer cell lines was higher than that in paracancerous normal tissues and normal bladder epithelial cells. Downregulation of
SPRY4-IT1 expression by siRNA interference significantly inhibited the proliferation and migration of bladder cancer cells and promoted apoptosis of bladder cancer cells [
23]. To date, little is known about the role of
LINC00612 in tumors. In this study, we found that
LINC00612 is significantly upregulated in bladder cancer tissues and cell lines. Further studies confirmed that
LINC00612 could promote tumor proliferation and invasion in vivo and in vitro, suggesting that
LINC00612 may be a potential target for observation and treatment of bladder cancer.
Researchers at Harvard University proposed the ceRNA hypothesis in 2011, arguing that there is a pattern of interactions between miRNA and mRNA. The hypothesis further states that various types of RNA molecules (including mRNA, lncRNA, etc.) can regulate each other by competitively inhibiting miRNAs via common microRNA response elements (MRE) [
24]. The stability and transcription of cytoplasmic lncRNA can be modified by trapping miRNAs, thereby altering signaling pathways. In this study, we found that
LINC00612 was primarily localized in the membrane, which indicated that the ceRNA mechanism may exist. Subsequently, bioinformatic predictions, luciferase reporter gene experiments, RIP experiments, and RNA pull-down experiments revealed that
LINC00612 directly binds and sponges
miR-590.
miR-590 has been shown to play an important role in a variety of cancers, and its function has been described as being carcinostatic in breast cancer, osteosarcoma and lung cancer [
25‐
28]. However, some studies reached different conclusions. These studies suggested that
miR-590 could promote tumor cell proliferation and enhance tolerance to radiotherapy [
6‐
8]. The effect of
miR-590 in bladder cancer remains unclear. In this study, we showed that
miR-590 could be sponged by
LINC00612 and therefore counter the carcinogenic effect of
LINC00612, which provides a basis for confirming the carcinostatic effect of
miR-590 in bladder cancer. Agostino [
29] suggested that
miR-590 can be specifically adsorbed by the lncRNA
MIR205HG in head and neck squamous cell carcinomas, thereby leading to uncontrolled tumor cell proliferation. This ceRNA regulation mechanism was similar to our findings.
EMT is a biological process in which epithelial-derived malignant cells transform into mesenchymal cells with increased migration and invasion ability [
30]. The characteristic changes in EMT include the loss of polarity of epithelial cells, degradation of intercellular junctions, changes in cell morphology due to the reorganization of cytoskeletal structures, and downregulation of epithelial gene expression accompanied by upregulation of mesenchymal gene expression. These changes provide the cell with a greater ability to migrate, invade and degrade the extracellular matrix [
31]. During EMT, cells lose epithelial marker factors, such as E-cadherin, while mesenchymal markers increase, such as vimentin, N-cadherin, and fibronectin, and related transcription factors, including Twist, Snail, and Zeb families, are activated. The results of this study conclude that
LINC00612 promotes EMT in BC cells by inhibiting the expression of the epithelial marker E-cadherin and by enhancing the expression of the mesenchymal marker vimentin, thus increasing the proliferation and invasion of BC cells. This regulation was competitively adjusted by
miR-590, according to the ceRNA mechanism.
miR-590 has been previously reported as being an EMT inhibitory miRNA [
32], which is in accordance with our findings. Furthermore, we demonstrated that
miR-590 could directly bind to downstream
PHF14 at the 3’UTR.
PHF14 is involved in several signaling pathway, including the classical TGF-β signaling pathway [
33]. Several researchers have demonstrated that
PHF14 is overexpressed in biliary tract cancer and lung cancer and may also be involved in tumorigenesis [
34,
35]. To measure whether the regulation of the
LINC00612/
miR-590/PHF14 axis modulated cellular EMT and thus modulated the proliferation and invasion of BC cells, rescue experiments in vitro and in vivo were conducted. When
LINC00612/
miR-590 and
LINC00612/
shPHF14 (
shLINC00612/
PHF14) were cotransfected into BC cells, the alterations in cellular EMT, cell proliferation and invasion were restored. These results confirmed that the
LINC00612/
miR-590/
PHF14 axis had a substantial effect on BC cellular EMT and might be a crucial modulator of cell proliferation and invasion in BC cells. At present, the function and regulation of
LINC00612 in other tumors remains unclear. Meanwhile, the regulation of the
LINC00612/
miR-590/
PHF14 axis in bladder cancer requires confirmation in large-scale clinical research studies, which will be the main aim of a future study.