Background
Bladder cancer is the most common cancer of urinary system in china. Although approximately 70 % of patients will preliminary diagnosis as nonmuscle-invasive BC (NMIBC), 50–70 % of patients will recur and about 10–20 % will progressed to muscle-invasive BC (MIBC) [
1]. So, exploring the early diagnostic and prognostic markers for bladder cancer and molecular mechanisms involving in bladder cancer is significant for raising the survival rates of bladder cancer patients. Nowadays, many mechanisms involving in bladder cancer have been confirm by studies, such as the activation of proto-oncogene, the inactivation of tumor suppressor gene (point mutation, rearrangement and deficiency), chromosome abnormality and so on. Since many mechanisms are still unclear, there is a need to further understand the molecular mechanisms involving in bladder cancer development for exploring the effective therapeutic modalities and early detection approach.
The long non-coding RNA (lncRNA) is a kind of RNA with size over 200 nt and has no protein-coding capacity [
2]. Unlike classical coding genes, which function by translated into protein molecules, lncRNAs play a key roles in regulation of various biological process in the shape of RNA and have exhibited less evolutionary constraint [
3,
4]. The expression of genes has been revealed to be regulated by lncRNAs in kinds of different approaches including repression of neighboring (
cis) genes, distant (
trans) via histone modification, and through interaction with miRNAs [
5‐
7]. Increasing evidences have indicated that lncRNAs were closely involved in carcinogenesis and have the potential to be early tumor diagnostic markers and molecular-targeted therapy sites [
5,
8,
9]. In BC, lncRNAs are associated with carcinogenesis, development and prognosis [
10,
11]. Xue et al. found that the low expression of lncRNA MDC1-AS was involved in BC by up-regulation of its antisense tumor suppressing gene MDC1 [
12]. He et al. revealed a new lncRNA, linc-UBC1 (Up-regulated in bladder cancer 1), was over-expressed in BC tissues and it was associated with lymph node metastasis and poor survival [
13]. Accumulating evidence indicated that abnormal expression of lncRNAs had a close relationship with cancers [
14,
15]. So, lncRNAs have the potential to be diagnostic markers and therapeutic targets for BC in the clinic.
It is generally accepted that lncRNAs, as any other protein-coding gene, undergo the same regulatory mechanisms including epigenetic regulation [
16,
17]. As one of the most extensively studied epigenetic change, Aberrant DNA methylation is associated with various biological processes including cancer [
18‐
21]. It is a procedure of chemical modification which will specifically methylate the cytosines located 5′ to guanosines in CpG dinucleotides and give rise to 5-methylcytosine (m5C) via the DNA methyltransferases (DNMTs) [
22]. As one of the DNMTs, DNMT1 has the power to maintain the methylation of newly replicated DNA. Studies have demonstrated that lncRNAs could associate with DNMT1, contributing to the expression of gene and aberrant DNA methylation during the tumorigenesis [
23]. However, whether the tumorigenesis and development of BC can affected by lncRNAs via DNMT1 or not and the molecular mechanism involved in the process are unclear.
Lately, studies had identified that lncRNA may directly associate with DNMT1 through binding to it, and prevent the methylation of tumor suppressor gene [
24]. In colon cancer, lncRNA with low expression was also found to regulate epigenetic modifications and the expression of specific gene by assembles DNMT1 at specific genomic sites [
25]. To examine if this function be suitable for BC, we investigated a well-studied tumor suppressor gene DBCCR1 (deleted in bladder cancer chromosome region 1) with a methylation sensitive and lncRNA DBCCR1-003 (name got from the database of lncRNAs, transcript ID:ENST00000482797) arising from the locus of DBCCR1. DBCCR1 is located at chromosome 9q32-33 identified by loss of heterozygosity (LOH) studies of human BC to act as a tumor suppressor gene [
26]. Performing demethylation experiments in BC cells resulted in the re-expression of DBCCR1 mRNA indicating that DBCCR1 expression is silenced by hypermethylation [
27]. These features make DBCCR1 be a good candidate for our study.
To prove our hypothesis, we first test the expression of DBCCR1-003, DBCCR1 and DNMT1 as well as methylation state of DBCCR1 promoter in BC cells and tissues. Then, we investigated the expression change of DBCCR1-003, DBCCR1 and DNMT1 and methylation dynamics of DBCCR1 by knock-in DBCCR1-003 and conducting demethylation treatment in BC cells. The function of DBCCR1-003 was determined by using cell proliferation, clone formation assay, cell apoptosis and cell cycle analysis. RNA immunoprecipitation (RIP) was conducted to confirm if DBCCR1-003 physically associates with DNMT1. Chromatin immunoprecipitation (ChIP) was performed to measure the binding of DNMT1 in DBCCR1 CpG island promoter. According to the research conclusions above and our previous results, this study was designed to detect that whether the tumorigenesis and development of BC can affected by lncRNA DBCCR1-003 via DNMT1 or not, and investigate the possible underlying molecular mechanism involved in the process.
Methods
Patients and tissue samples
This study has been reviewed by the Institutional Review Board (IRB) of Guangzhou Medical University with the approval number of GMU-IRB#: 2015-11. After being acquainted with the aim and the methods used in the study, each of the patients included in the study signed a written informed consent form. Between January 2012 and May 2015, a total of 24 specimens were obtained from patients undergoing BC surgery at the first affiliated Hospital of Guangzhou medical University (Kangda Road 1#, Haizhu District, Guangzhou, Guangdong, China). None received any antitumoral treatment prior to tumour sampling. All specimens were pathologically graded and staged according to the TNM and World Health Organization classification. The histopathological classification of urinary BC was confirmed by two independent histopathologists. A total of 24 adjacent tissues of cancer from matched patients were collected as control group. The patients included 14 males and 10 females. The median patient age was 69 years with range 47 to 90 years. More details of characteristics are classified into Table
1.
Table 1
Clinicopathologic features of BC patients and the levels of DBCCR1-003 and DNMT1 expression in the cancer tissues
All patients | 24 (100 %) | | | | |
Gendera
| | | 0.403 | | 0.639 |
Females | 10 (41.6 %) | 4.61 (1.10–4.19) | | 1.68 (1.2–2.39) | |
Males | 14 (58.4 %) | 1.60 (0.56–3.70) | | 1.87 (0.65–3.43) | |
Age at diagnosisa
| | | 0.887 | | 0.931 |
≤70 years | 12 (50.0 %) | 2.0 (0.58–3.70) | | 1.85 (0.65–3.43) | |
>70 years | 12 (50.0 %) | 1.91 (0.56–4.19) | | 1.74 (0.97–2.39) | |
Pathologic gradea
| | | 0.02* | | 0.035* |
Low grade | 8 (33.3 %) | 2.90 (1.39–4.19) | | 1.4 (0.97–2.19) | |
High grade | 16 (66.7 %) | 1.39 (0.57–2.81) | | 1.98 (1.15–3.43) | |
Pathologic stageb
| | | 0.14 | | 0.037* |
pTa | 5 (20.8 %) | 3.39 (2.51–4.19) | | 1.26 (0.65–1.84) | |
pT1 | 13 (54.2 %) | 1.84 (0.66–3.70) | | 1.74 (1.0–2.39) | |
≥pT2 | 6 (25.0 %) | 1.02 (0.57–1.57) | | 2.34 (1.35–2.71) | |
Cell culture and transfection
Both of the human urinary bladder transitional carcinoma cell lines T24 and human bladder epithelial immortalized cell lines SV-HUC-1 were purchased from American type culture collection (ATCC). The T24 cells was cultured in RPMI 1640, and SV-HUC-1 cells was cultured in F12K. All medium were supplemented with 10 % fetal bovine serum (Gibco, USA), in a humidified air atmosphere of 5 % CO2 at 37 °C. We had used 0.25 % trypsin (with 1 mM EDTA) (Invitrogen, Carlsbad, CA) to harvest the cells for further experiment. Cells were grown in polystyrene 25 cm2 dishes and transfected with 3.0 μg of DNA using 30 μl of Lipofectamine transfection reagent (Life Technologies) according to the manufacturer’s recommendations for 6 h.
Construction of vectors
Plasmid cDNA-DBCCR1-003 was constructed by introducing SpeI-NotI fragment containing the DBCCR1-003 cDNA into the same site in LentiORF PLEX-MCS vector. The recombinant vectors were designated as LentiORF PLEX-MCS-DBCCR1-003 and identified by sequencing. At the same time, we also constructed the control vector named PLEX-MCS-control. Both of the vectors were transfected in 293FT cells, respectively. Generated virus particles subsequently infected T24 cells, the positive clones were obtained following puromycin selection. The stable cell lines achieved were correspondingly designated as Lenti-DBCCR1-003(L-D3) and control Lenti-vector (L-C).
Real-time quantitative PCR
Total RNA was extracted using the Trizol Reagent (Invitrogen, USA) according to the instructions. The RNA purity and concentration were determined by the UV spectrophotometer. cDNA was reversibly transcribed from the extracted total RNA using an MMLV reagent kit (TaKaRa, Japan) and the primers were designed as Additional file
1: Table S1. The expression of the filtered lncRNAs and their associated encoding genes was measured using SYBR real-time PCR (qPCR) (Takara Bio, Otsu, Japan) according to the manufacturer’s instructions. PCR was then carried out as follows: denaturing at 95 °C for 20 s, 40 cycles of 10 s at 95 °C, 20 s at 58 °C and 30 s at 72 °C.
5-Aza-2′-deoxycytidine (DAC) treatment
T24 cells were seeded at 50 % confluence 6 h before treatment. The doses of DAC (Sigma, St Louis, MO) for T24 cells were 12.5 μmol/l. The cell was treated with the designated doses for 48 h, and the confluence of the collected cell was never greater than 80 %.
Western blot analysis
Cultured cells were collected and washed three with 1 ml of PBS. After cracked with protein lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 %Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin), cells were collected in a centrifuge tube. Cell lysates were centrifuged at 13,000×g for 15 min at 4 °C and insoluble debris was discarded. Soluble proteins were subjected to 8 % SDS-PAGE, after electrophoresis, the proteins were transferred onto PVDF membrane and detected by immunolabeling with primary and secondary anti-bodies. In this experiment, we made GAPDH as the internal reference. Protein bands were quantified using Chemiluminescence with Koda film.
Methylation-specific PCR (MSP)
Primer sequences for DBCCR1 were list in Additional file
1: Table S1. Genomic DNA of tissues were extracted from frozen specimens and digested by proteinase K followed by standard phenol/chloroform purification and ethanol precipitation. Reagents required for the bisulfite modification of DNA were supplied in the EZ DNA Methylation-Gold Kit (ZYMO RESEARCH). The process was performed according to the manufacturer’s recommendations. 1 μg of DNA was modified with sodium bisulfite to convert all unmethylated (but not methylated) cytosine to uracil followed by amplification with primers specific for methylated versus unmethylated DNA [
28]. DNA from normal lymphocytes was used as control. Water was also used as negative control for contamination. Methylation status of each tumor was evaluated in triplicate for reproducible in MSP. PCR products were electrophoresed on a 2 % agarose gel for analysis.
Cell proliferation analysis
Cells were plated into a new dish. 1 × 105 cells were plated in triplicate and harvested at the indicated time points: 24, 48, 72, and 96 h. The number of cells was determined using an Auto T4 Plus Cell Counter (Nexcelom Bioscience, USA). Triplicate plates were counted for each cell lines.
Cell survival was measured using a standard colony forming assay. Cells were seeded onto six-well plates at 400 cells per well. One week later, colonies were fixed with 100 % methanol for 15 min and stained with 0.1 % crystal violet for 20 min. Microscopic colonies composed of more than approximately 50 cells were counted as having grown from surviving cells.
Apoptosis determination by flow cytometry
The cells were harvested by centrifugation for 3 min at 1000 rpm and were resuspended in binding buffer. Aliquots containing 1 × 105 cells in 190 μl of buffer were stained with 10 μl of PI solution and with 5 μl of Annexin V-FITC (eBioscience, USA) for 10 min at room temperature. The excitation and emission wavelengths of FITC was FL1 PMT with 515–545 nm, and PI was FL3 with 650 nm. Then Flow cytometric analysis was performed using a flow cytometer (BD, USA) to detect the cell apoptosis.
Cell cycle analysis
Cells were collected by trypsin method, washed with PBS, fixed overnight at 4 °C in 70 % ethanol. They were then washed in cold PBS and resuspended in 50 μg/ml propidium iodide and RNase A (50 μg/ml). The cell suspension was incubated in a 37 °C water bath for 1 h and cell cycle distribution was determined by flow cytometry. The cell cycle phase quantification was performed using ModFit LT to detect the cell apoptosis.
Chromatin immunoprecipitation (ChIP) assay
Chromatin immunoprecipitation was performed with EZ-Magna ChIP A/G kit (Millipore) according to manufacturer’s instructions. Briefly, protein extract form 1 × 10
7 cells were used for each reaction. Proteins were cross-linked to DNA by adding formaldehyde to the cell culture medium to a final concentration of 1 % at room temperature for 10 min and quenched by addition of 0.125 M glycine for 5 min at room temperature. The nucleus was isolated with nuclear lysis buffer (Millipore) supplemented with protease inhibitor cocktail (Millipore). Cells were sonicated and sheared to yield fragments between 200 and 1000 bp. 5 μg of either anti-DNMT1 (Abcam), Normal mouse IgG (the negative control) and anti-RNA polII (Millipore) (the positive control) was added to the sonicated samples and incubated at 4 °C overnight with rotation. Immune complexes were collected with Protein A/G agarose beads and washed with low salt buffer, high salt buffer, LiCl buffer and TE buffer to remove nonspecific binding. Protein/DNA complex was reverse cross-linked and DNA was purified using spin columns. Purified DNA was detected with quantitative PCR. Primers for ChIP-qPCR were listed in Additional file
1: Table S1.
RNA immunoprecipitation (RIP) assay
RIP was conducted as described in [
49]. Briefly, 1 × 10
7 cells were harvested and lysed in complete RIP lysis buffer. Resuspended nuclear fraction was sheared by a homogenizer and sonicated. Antibodies against DNMT1 (Abcam) and IgG (Sigma-Aldrich) were incubated with magnetic beads (Protein A or G) for 1 h and the nuclear lysates were incubated overnight with rotation. Samples were incubated with Proteinase K and dealed with QIAamp MinElut Virus Spin kit (QIAGEN, GER) to isolated RNA. Purified RNA was reverse transcribed into cDNA by random primer (GeneCopoeia, USA), and detected with quantitative PCR. The binding of ecCEBPA and DNMT1 which had been identified by studies was used as a positive control [
24], and normal mouse IgG was used as a negative control. Primers for RIP-qPCR were listed in Additional file
1: Table S1.
Statistical analysis
All quantified data were analyzed by the SPSS 13 statistical software. Statistical significance was measured by Student’s t test and Mann–Whitney U-test. The relationship between the expression level of DBCCR1-003 and clinicopathologic parameters were analyzed using the Mann–Whitney U-test when comparing the differences between two groups, and using the Kruskal–Wallis test when comparing the differences among three or more groups. All p values <0.05 were considered significant.
Discussion
Nowadays, there have been raising interests in the role of lncRNAs in human diseases, especially when it involves in the epigenetic modifications. An accumulating number of studies identified that the epigenetic dysregulation of lncRNAs expression in cancer play a key role [
7,
17]. In this study, our results indicate that down-regulation of DBCCR1-003 in BC is responsible for the down-regulation of DBCCR1 via DNMT1, and overexpression of DBCCR1-003 can inhibit cell growth by inducing apoptosis and arresting the cell cycle in phase in T24 cells, revealing a new lncRNA DBCCR1-003 which can affect the tumorigenesis and development of BC by mediating tumor suppress gene DBCCR1 via DNMT1.
LncRNAs belong to a versatile group of RNA transcripts without protein-coding potential that function via diverse mechanisms and act as regulator in important biological processes [
34]. Taking into account the widespread function that lncRNAs act in cellular networks, there is no surprising that lncRNAs have been involved in human diseases, including the cancer [
35]. Studies have indicated that various lncRNAs are related to cellular transformation, having the potential to be tumor suppressors or oncogenes, and leading to tumorigenesis [
36]. In gastric cancer, a newly identified lncRNA, CARLo-5, was found to be up-regulated and its knock-down significantly inhibited the cell proliferation [
37]. Maternally Expressed Gene 3(MEG3), an imprinted gene that encodes a lncRNA, lost its expression and was negatively associated with tumorigenesis in BC [
38]. We found the low expression of DBCCR1-003 in T24 cells and BC tissues, and the cell growth of T24 cells was inhibited by increasing the expression level of DBCCR1-003, indicating that DBCCR1-003 plays a key role in inhibiting BC growth. To explore the possible mechanism responsible for the growth inhibition effect of DBCCR1-003, we performed flow cytometry assay, and found that knock-in DBCCR1-003 induced G0/G1 cell-cycle arrest and cell apoptosis in T24 cells, indicating that BC cell growth mediated by DBCCR1-003 may be related to the regulation of cell cycle and apoptosis. Similar to our results, Shi et al. reported that overexpression of lncRNA, BRAF activated non-coding RNA(BANCR), would suppress colorectal cancer cell growth in vitro and in vivo which was related to induction of G0/G1 cell cycle arrest and apoptosis by regulating p21 [
39]. Ma et al. revealed that lncRNA-LET overexpression conferred a inhibition to the cell growth of gallbladder cancer cells through promotion of cell cycle arrest at G0/G1 phase and to the induction of apoptosis under hypoxic conditions [
40]. Collectively, our results confirm the tumor-suppressive activity of DBCCR1-003 and suggest that overexpression of DBCCR1-003 inhibits BC growth through the inducing G0/G1 arrest and apoptosis.
In gallbladder cancer, the down-regulation of lncRNA-LET was observed to be associated with poor prognosis, higher tumor status, nodal status, and clinical stage [
41]. The similar result was also found in colorectal cancer that lower expression of lncRNA BANCR was related to increased tumor sizes [
41]. To DNMT1, DNA hypermethylation on CpG islands is related to the overexpression of DNMT1 in multistage of BC [
42]. Consistent with these studies, our results demonstrated that there is the down-regulation of DBCCR1-003 and up-regulation of DNMT1 is related to BC grade and stage.
It has been identified that the dysregulation of lncRNAs is associated with cancer epigenetics [
42]. Besides the known and possible epigenetic mechanisms that the lncRNAs involved in cancer can act on tumor suppressor genes, they may associate with a DNA methyltransferase and recruit it to the promoter region of a tumor suppressor gene, leading to the transcriptional silencing of the latter [
43]. For example, Li et al. revealed that a lncRNA named AS1DHRS4 (antisense 1 dehydrogenase/reductase SDR family member 4), transcribed from the locus of the DHRS4 gene known to be involved in cancer, modulated the expression of DHRS4 by epigenetic regulation at the DHRS4L2 promoter region [
44]. Based on these facts, we hypothesizes that DBCCR1-003 may act as a tumor suppressor gene through regulating the expression of DBCCR1 via DNA methylation. To prove that, we first determined the expression and methylation status of DBCCR1 and found that, like the DBCCR1-003, it was down-regulated and hypermethylation in T24 cells and BC tissues. Then, we knock-in DBCCR1-003 in T24 cells and found that the expression of DBCCR1 was also increased whereas the methylation level of DBCCR1 was decreased. Moreover, treating with DAC in T24 cells, DBCCR1 was up-regulated while the methylation level of DBCCR1’s CpG island was decreased. Meanwhile, similar to the effect of knock-in DBCCR1-003, the cells growth was inhibited and G0/G1 cell-cycle arrest and cell apoptosis was induced in T24 cells treated with DAC. Taken together, these results suggest that DBCCR1-003 may act as a tumor suppressor gene through regulating the expression of DBCCR1 via DNA methylation.
Epigenetic dysregulation of cellular genes is an important feature in the development of human malignancy. More and more evidence has suggested that DNA methylation is among the key players in the human urinary system tumors. The DNA methylation is catalyzed by DNMTs. As one of the three main types of DNMTs (DNMT1,DNMT3A, and DNMT3B)involved in genomic DNA methylation, DNMT1 displays a obvious favour for hemimethylated over unmethylated DNA and maintains DNA methylation [
45]. Deepika Dhawan and his coworkers have found that DNMT1 has excellent potential as a target for invasive urothelial carcinoma therapy in human being [
46]. The high expression of DNMT1 was demonstrated to play a key role in cell transformation in vitro, suggesting that the abnormal expression of DNMT1 may have influence to the development of human cancer [
47]. In our study, we found that the upregulation of DNMT1 expression in T24 cells and BC tissues was related to DBCCR1 low expression with hypermethylation of DBCCR1 promoter. The upregulation of DBCCR1 expression is associated with a decrease of DNA methylation of DBCCR1. Intriguingly, the ChIP assay also identified that the binding of DNMT1 in DBCCR1 CpG island promoter were obviously decreased. These results suggested that DNMT1 up-regulation is responsible for the hypermethylation of DBCCR1. In addition, Wang et al. identified that a novel lncRNA Dum, which modulated DNA methylation by recruiting Dnmts to specific promoter regions, silencing its neighboring gene Dppa2 [
48]. Chalei et al. reported that multiple genes could be modulated by a lncRNA Dum to stay away from their site of synthesis through binding to DNMT1 and changing DNA methylation status [
49]. Similar to these studies, we found the expression of DNMT1 did not change when DBCCR1-003 was overexpressed. Performing the RIP assay to determine if DBCCR1-003 physically associates with DNMT1 in T24 cells, the results indicated that DBCCR1-003 may bind to DNMT1 in T24 cells. Collectively, the above studies and our results suggest that the expression of DBCCR1 may be regulated by DBCCR1-003 via binding to DNMT1 without affecting the expression of DNMT1 and preventing DNMT1-mediated the methylation of DBCCR1 in BC.
Authors’ contributions
Conceived and designed the experiments: JW, QD. Performed the experiments: QD, LJ,QB, CZ, YM. Analyzed the data: LJ, LM, ZG. Contributed reagents/materials/analysis tools: SJ, QB. Wrote the paper: LJ, JW. All authors read and approved the final manuscript.