MicroRNA-218 inhibits bladder cancer cell proliferation, migration, and invasion by targeting BMI-1
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MicroRNAs (miRNAs) are recognized as important molecules and have emerged as important gene regulators in tumorigenesis. Growing evidence suggested that miR-218 was a tumor suppressor in many human cancers. However, its underlying role in bladder cancer (BCa) remains unclear. The aim of this study was to explore the effect of miR-218 on the proliferation, migration, and invasion of BCa cells. We found that miR-218 was frequently downregulated in BCa tissues compared with normal adjacent tissues. In vitro and in vivo assays demonstrated that miR-218 overexpression in the BCa cells inhibited cell proliferation, migration, and invasion. Luciferase reporter assay showed that BMI-1 was a direct target of miR-218. In addition, we found that miR-218 regulated the expression of BMI-1 and its downstream target (PTEN) and participated in the phosphorylation of AKT. Our findings indicate that miR-218 functions as tumor suppressor in BCa, and the miR-218/BMI-1 axis may provide novel diagnostic and therapeutic strategies for the treatment of BCa.
KeywordsMicroRNA-218 Bladder cancer BMI-1 Proliferation Migration Invasion
Bladder cancer (BCa) is among the most common urological cancers, with more than 330,000 new cases each year and resulting in more than 130,000 deaths per year . BCa can be classified into two categories: nonmuscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). The 5-year survival rate for patients with NMIBC is close to 90 %, whereas that of patients with MIBC is only approximately 60 % . Thus, an improved and detailed understanding of the mechanisms underlying BCa pathogenesis is urgently needed. Presently, growing evidence suggests that microRNAs (miRNAs or miRs) play an important role in BCa pathogenesis, thus providing new opportunities for the treatment of this disease.
miRNAs are short noncoding RNAs of approximately 22 nucleotides in length that regulate protein-coding gene expression by pairing with the 3′-untranslated regions (3′-UTR) of specific target messenger RNA (mRNA) . Some miRNAs expressed at high levels might function as tumor oncogenes; conversely, miRNAs expressed at low levels might function as tumor suppressors in human cancers . Recently, some research has shown that miR-218 is downregulated and functions as a tumor suppressor in many human cancers, including renal cell carcinoma, gastric cancer, and nasopharyngeal cancer [5, 6, 7]. Additionally, a recent study by Tatarano et al. identified miR-218 as a tumor suppressive miRNA in BCa . However, the role of miR-218 in BCa has not been studied in depth.
In this study, we compared the miR-218 expression levels in BCa tissues and normal adjacent tissues (NATs). We then studied the effects of miR-218 overexpression in BCa cell lines T24 and EJ on cell proliferation, migration, and invasion. In addition, B lymphoma mouse Moloney leukemia virus insertion region 1 (BMI-1) was considered and validated as a target gene of miR-218. Altogether, our data contributes to elucidate the role of miR-218 in BCa.
Materials and methods
Clinical specimens and cell culture
Twenty-seven pairs of BCa tissues and NATs were obtained from BCa patients who underwent radical cystectomy at the First Affiliated Hospital of Nanjing Medical University, China. All patients provided signed informed consent, and this study was approved by the Research Ethics Committee of our institution. The specimens were immediately frozen and stored in liquid nitrogen until total RNA extraction. Patient information and clinical pathological data are summarized in Table S1.
The human BCa cell lines T24 and EJ were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS; Gibco, Australia) and 1 % penicillin-streptomycin in an incubator with humidified 5 % CO2 at 37 °C.
Establishment of miR-218 overexpressing cells
Lentivirus overexpressing miR-218 and negative control lentivirus were constructed as previously described  and transfected into T24 and EJ cells, according to the manufacturer’s instructions. Since the vector contains a puromycin resistance gene and the green fluorescent protein gene, all transfected cells were treated with 2 μg/ml puromycin for 24 h to select the stable cell lines. Fluorescence microscopy and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) were applied to determine the efficiency of transduction.
RNA extraction and qRT-PCR
Total RNA, including miRNA from the 54 frozen bladder tissues and the transfected cells, was extracted using miRNeasy Mini Kit (Qiagen). Extracted RNA was prepared using an All-in-One™ miRNA qRT-PCR Detection Kit (GeneCopoeia, FulenGen, China), according to the manufacturer’s instructions. The expression of BMI-1 was determined using SYBR Green PCR Kit (Takara, Japan). U6 and β-actin RNAs were used as internal controls for miRNA and mRNA detection, respectively. The validated miRNA-specific forward primer (GeneCopoeia, FulenGen, China) was used for the qRT-PCR assay. The primers for qPCR were obtained from Sango Biotech (Shanghai, China), and the sequences were as follows: BMI-1 (forward: 5′-GTG CTT TGT GGA GGG TAC TTC AT-3′, reverse: 5′-TTG GAC ATC ACA AAT AGG ACA ATA CTT-3′) and β-actin (forward: 5′-AGC GAG CAT CCC CCA AAG TT-3′, reverse: 5′-GGG CAC GAA GGC TCA TCA TT-3′).
Cell viability assay and colony formation assay
For the cell viability assay, the transfected cells were seeded into 96-well plates at a density of 3000 cells per well. The CCK-8 method was used to determine cell viability at 0, 24, 48, and 72 h after the cells were seeded. The absorbance was measured at 450 nm using a Tecan Infinite F200 microplate reader.
For the colony formation assay, the transfected cells were seeded into six-well plates at a density of 200 cells per well and maintained in RMPI 1640 containing 10 % FBS for 2 weeks. The colonies were imaged and quantified after fixing with methanol and staining with 0.1 % crystal violet.
Wound healing assay
The transfected cells were seeded into six-well plates and cultured until they reached 95 % confluence. A 200-μL pipette tip was used to generate a cross-shaped wound through the center of the well. The cultures were washed with phosphate-buffered saline (PBS) to remove cell debris, and then the cells were incubated in RPMI 1640 medium containing 10 % FBS. The wound was observed under a microscope (Olympus, Japan) at ×40 magnification at two preselected time points (0 and 24 h), and the widths of wounds were counted.
Migration and invasion assays
Twenty-four-well transwell chambers with an 8.0-μm pore size polycarbonate membrane were used to measure cell migration and invasion. For the invasion assay, the upper surface of the membrane was coated with BD Matrigel (BD Biosciences, USA) at 37 °C for 4 h, whereas for the migration assay, the top chamber was not coated with BD Matrigel. A total of 2 × 104 cells were suspended in 200 μL of serum-free medium and seeded on the upper chamber. Medium containing 10 % FBS was added to the bottom chamber as a chemoattractant. The cells were incubated at 37 °C for 48 h for the invasion assay or 24 h for the migration assay. The cells in the top chamber were removed with cotton swabs. The cells on the lower membrane surface were fixed with methanol, stained with 0.1 % crystal violet, and counted under a microscope at ×100 magnification (Olympus, Japan).
Western blot analysis
The transfected cells were lysed, and the protein was extracted using RIPA buffer (Beyotime, China) and quantified by a BCA Protein Assay Kit (Beyotime, China). Equivalent quantities of protein were separated on 10 % SDS–PAGE gels and transferred to polyvinylidene fluoride membranes. After blocking with 5 % nonfat milk at room temperature for 1 h, the membranes were immunostained with primary antibodies at 4 °C overnight, washed three times in TBST, and then incubated with secondary antibody at room temperature for 1 h. Band signals were detected using a chemiluminescence system (Bio-Rad, USA) and analyzed using Image Lab Software. The following primary antibodies were used: BMI-1 (Cell Signaling Technology, 1:1000), PTEN (Cell Signaling Technology, 1:1000), AKT (Cell Signaling Technology, 1:1000), and phospho-AKT (Cell Signaling Technology, 1:1000). The protein levels were normalized to GAPDH (Cell Signaling Technology, 1:1000).
The expression of BMI-1 protein was also assessed by immunofluorescence. The transfected cells grown on coverslips were fixed with 4 % paraformaldehyde at 4 °C for 10 min, permeabilized with 0.1 % Triton X-100 in PBS for 5 min at room temperature, and blocked with 5 % BSA for 1 h at room temperature. Next, the cells were incubated with rabbit anti-BMI-1 (Cell Signaling Technology, 1:500) at 4 °C overnight, followed by incubation with goat anti-rabbit IgG-FITC (Cell Signaling Technology, 1:600) for 1 h at room temperature. To visualize the nucleus, the coverslips were counterstained with 4′6-diamidino-2-phenylindole (DAPI). Fluorescent images were captured by a confocal laser microscope (Olympus, Japan).
Luciferase reporter assay
For the luciferase reporter assay, nontransfected T24 cells were cultured in 24-well plates and then cotransfected with plasmid containing pEZX/BMI-1-3′-UTR or pEZX/BMI-1-3′-UTR-mutant together with firefly and renilla luciferase, and miR-218 mimics or control, using Lipofectamine 2000 (Invitrogen, USA). Firefly and renilla luciferase activities were measured 24 h after transfection using the Luc-Pair miR Luciferase Assay (GeneCopoeia, FulenGen, China). Normalized data were calculated as the ratio of luminescence from firefly to renilla luciferase.
In vivo tumor xenograft studies
The transfected T24 cells and controls were implanted with 5 × 106 cells per site bilaterally on the buttocks in Balb/c nude mice (4 weeks old, n = 6). Tumor growth was monitored by measuring the width (W) and length (L) with calipers every 3 days, and the volume (V) of the tumor was calculated using the formula V = (W 2 × L) / 2. At the end of the experiment, the tumors were removed and fixed in 4 % formalin for immunohistochemical analysis. The animal studies were performed in accordance with the institutional ethics guidelines for animal experiments.
Hematoxylin and eosin (H&E) staining and immunohistochemical staining
Formalin-fixed tumor tissues from nude mice were embedded in paraffin and cut into thin sections. The tissue sections were stained with H&E using standard procedures. For immunohistochemical staining, tissue sections were dewaxed and then rehydrated in graded ethanol. Antigen retrieval was performed by microwave heating in sodium citrate buffer (pH 6.0). The sections were immersed in 3 % H2O2 for 10 min to block endogenous peroxidase activity and then incubated with Ki-67 primary antibody (Cell Signaling Technology, 1:400) at 4 °C overnight. After washing, the sections were incubated with HRP-conjugated secondary antibody at room temperature for 1 h, and reactive products were visualized by staining with 3,3′-diaminobenzidene (DAB). The images were obtained under a microscope (Olympus, Japan) with appropriate magnification.
Statistical analyses were performed using SPSS 13.0 and GraphPad Prism 5. All data are presented as the mean ± standard deviation. Differences between two groups were analyzed using the Student’s t test. The cell viability assay and in vivo tumor xenograft results were assessed by repeated-measures analysis of variance. A P value <0.05 was considered statistically significant. All experiments were repeated more than three times, and each experiment was performed in triplicate.
Expression of miR-218 was downregulated in BCa clinical specimens
Establishment of stable BCa cell lines T24 and EJ overexpressing miR-218
Our results demonstrated that miR-218 was strongly downregulated in BCa specimens. Furthermore, several studies have confirmed that miR-218 has tumor-suppressive effects in glioma and colon cancer cells [10, 11]. Therefore, we hypothesized that miR-218 overexpression may have a similar effect in BCa cells. To test this hypothesis, we first established the stable BCa cell lines T24 and EJ overexpressing miR-218, which were confirmed by qRT-PCR. The expression of miR-218 was approximately 200 times higher in miR-218 overexpressing cells than that in miR-control cells (Fig. 1b).
Overexpression of miR-218 inhibits BCa cell proliferation
Overexpression of miR-218 inhibits BCa cell invasion and migration
To explore the role of miR-218 in BCa cells, we performed a wound healing assay to investigate the effect of miR-218 overexpression on cell migration. These results suggested that miR-218 overexpression could significantly inhibit the mobility of BCa T24 and EJ cells (Fig. 2d, e). Furthermore, a transwell assay showed that miR-218 overexpression resulted in a significant decrease in the migratory and invasive capabilities of miR-218 overexpressing cells compared to the miR-control cells (Fig. 2f, g). These findings indicated that miR-218 overexpression had antimigratory and anti-invasive effects on BCa cells.
miR-218 directly targets the BMI-1 3′-UTR and negatively regulates its expression
Overexpression of miR-218 inhibits the growth of tumor xenografts in nude mice
To date, increasing evidence has demonstrated that aberrant expression of miRNAs can function as oncogenes by repressing tumor suppressor genes or act as tumor suppressors by negatively regulating oncogenes . Subsequently, changes in miRNA expression levels have been found to be associated with progression, metastasis, and recurrence of human cancers . Recently, studies have shown that some miRNAs, such as miR-145  and miR-26a , regulate BCa biological behaviors. As one of the miRNAs implicated in tumorigenesis, miR-218 has presented with a suppressive role during tumor progression. It has been reported that miR-218 serves as a tumor suppressor in numerous human cancers, including oral cancer , nasopharyngeal cancer , and gastric cancer . Furthermore, miR-218 overexpression in cancer cells has been shown to inhibit cell proliferation, invasion, and migration [17, 18]. Although it is known that miR-218 functions as a tumor suppressor, its molecular mechanisms in BCa are still unclear.
In the present study, we demonstrated that miR-218 was frequently downregulated in BCa tissues compared to NATs. To further assess the role of miR-218 in BCa in more detail, we first established the stable BCa cell lines T24 and EJ overexpressing miR-218. Next, we studied the gain-of-function effects of miR-218 on various aspects of T24 and EJ cells biology in vitro, which showed that miR-218 overexpression inhibits BCa cell proliferation, migration, and invasion. In the animal study, our findings were consistent with the effects of miR-218 overexpression in vitro and indicated that miR-218 inhibited tumor growth. Taken together, both the in vitro and in vivo studies suggested that the functional role of miR-218 in BCa pathogenesis might be as a tumor suppressor. Thus, the detailed mechanisms of miR-218 and its targets are worthy of further study.
Bioinformatics analysis demonstrated that BMI-1 is a potential direct target of miR-218 in BCa. As a proto-oncogene, BMI-1 is a member of the polycomb group (PcG) of genes and is the first identified PcG transcriptional repressor. BMI-1 plays an important role in the initiation and development of cancer. Many studies have demonstrated that BMI-1 is an oncogene that can regulate cancer initiation  and cell transformation  as well as induce epithelial–mesenchymal transition [21, 22]. Moreover, a study by Wu et al. confirmed that knockdown of BMI-1 in human BCa T24 cells inhibits cell proliferation and invasion . In our study, the dual luciferase reporter assay confirmed that miR-218 directly targets BMI-1. Additionally, qRT-PCR, western blot, and immunofluorescence experiments suggested that overexpression of miR-218 significantly caused the downregulation of BMI-1 at both the mRNA and protein level in T24 and EJ cells. Our immunofluorescence assay showed that BMI-1 protein is located in the nucleus of BCa cells (Fig. 3d). Furthermore, others have confirmed that BMI-1 protein is localized in the cytoplasm of noncancerous cells . So far, the nuclear-cytoplasm shuttling phenomenon remains to be determined. It is noteworthy that some of the PcG genes, such as NSPc1, may be associated with phosphorylation . As a PcG gene, BMI-1 has been demonstrated to regulate AKT phosphorylation in human cancers [26, 27].
Interestingly, Song et al. have reported that phosphatase and tensin homologue deleted on chromosome ten (PTEN) is a direct target of BMI-1 . In this study, we also found that the tumor suppressor PTEN was upregulated in miR-218-overexpressing cells in which BMI-1 was downregulated (Fig. 3b, c). Our findings are congruent with other research [28, 29]; that is, PTEN is a downstream target of BMI-1. Numerous studies have confirmed that PTEN as a suppressor of the PI3K/AKT signaling pathway participating in its regulation [30, 31]. These findings provide the molecular basis for activation of the PI3K/AKT signaling pathway in miR-218 overexpressing T24 and EJ cells. Furthermore, we confirmed that phosphorylation of AKT (pAKT) was suppressed in miR-218-overexpressing cells via the BMI-1/PTEN axis, whereas the total AKT level was unaffected (Fig. 3b, c). Taken together, all these data support the idea that miR-218 inhibits the proliferation, migration, and invasion of BCa, possibly via the miR-218/BMI-1/PTEN/AKT axis.
In conclusion, our study demonstrated that miR-218 is frequently downregulated in BCa tissues and functions as a tumor suppressor by directly targeting BMI-1. Thus, the miR-218/BMI-1 axis may provide novel diagnostic and therapeutic strategies for the treatment of BCa.
This work was supported by the Program for Development of Innovative Research Team of the First Affiliated Hospital of Nanjing Medical University, the Provincial Initiative Program for Excellency Disciplines of Jiangsu Province, the National Natural Science Foundation of China (Grant Nos. 81272832 and 81201997), the Natural Science Foundation of Jiangsu Province (Grant No. BK2011848), the Six Major Talent Peak Project of Jiangsu Province (Grant No. 2011-WS-121), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and Jiangsu Provincial Special Program of Medical Science (BL2012027). The funders had no role in study design, data collection, and analysis; decision to publish; or preparation of the manuscript.
Conflicts of interest
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