MicroRNA-892b influences proliferation, migration and invasion of bladder cancer cells by mediating the p19ARF/cyclin D1/CDK6 and Sp-1/MMP-9 pathways

  • Authors:
    • Seung-Shick Shin
    • Sung-Soo Park
    • Byungdoo Hwang
    • Bokyung Moon
    • Won Tae Kim
    • Wun-Jae Kim
    • Sung-Kwon Moon
  • View Affiliations

  • Published online on: August 25, 2016     https://doi.org/10.3892/or.2016.5052
  • Pages: 2313-2320
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Abstract

Cancers often utilize microRNAs to suppress tumor suppressor genes, thus facilitating their potential for growth and invasion. In the present study, we report the novel findings that miR-892b inhibits proliferation, migration and invasion of bladder cancer cells. The basal expression level of miR‑892b was significantly lower in 3 different bladder cancer cell lines than in normal human urothelial cells. Transfection of miR-892b mimics to bladder cancer cells resulted in dose‑dependent growth arrest. Flow cytometric analysis of the cell cycle showed that miR-892b-transfected bladder cancer cells were subject to arrest in the G1 phase, which was due to the downregulation of cyclin D1 and CDK6 followed by upregulation of p19ARF. In addition, overexpression of miR-892b impeded the migration and invasion of EJ cells. Expression of MMP-9 in EJ cells was blocked by transfection of miR-892b; the effect was regulated, at least in part, by activation of the Sp-1 transcription factor. Overall, we verified that miR-892b regulates the p19ARF/cyclin D1/CDK6 and Sp-1/MMP-9 signaling networks in bladder cancer cells and may provide a treatment option for advanced-stage bladder cancers.

Introduction

Bladder cancer is the fifth most common cancer, with a lifetime incidence rate of 1 in 26 for men in the US. The American Cancer Society stated that ~74,000 new cases were diagnosed in the year 2015; of these cases, ~16,000 were expected to die from the disease (1). The incidence of bladder cancer in males is 4 times higher than it is in females, and it is 2 times higher in white people than in black people for unknown reasons (1). The initiation and progression of bladder cancer is affected by various risk factors such as genetic alterations, and exposure to chemicals and carcinogens in cigarette smoke (1,2). Pathologically, transitional cell carcinoma (TCC) makes up ~90% of cases, and the other 10% consists of neoplastic lesions including squamous cell carcinoma, adenocarcinoma, sarcoma and small cell carcinoma. Although early detection followed by surgical resection extends the chances of survival, the 5-year survival rate for patients with advanced-stage bladder cancer is significantly less than 10% (1).

Bladder cells are transformed by a multistep process which includes genetic alterations such as chromosomal abnormalities (3), loss of tumor suppressor genes (4,5) and amplification of oncoproteins (6). Loss of tumor suppressor genes such as p53 or Rb is common in early stages, while amplification of oncoproteins is more common in advanced stages. However, the particular mutations that give rise to bladder cancer are not yet clearly understood, and even the mutational status of tumor suppressors is unclear. Despite its high impact on public health, there have been relatively few studies on the mechanisms of tumorigenesis of bladder cancer compared to other types of cancers. Therefore, it is critically important to identify molecular targets in order to find novel therapeutic options for this deadly disease.

MicroRNAs (miRs) are small non-coding RNA fragments that regulate gene expression by binding to the 3′-untranslated regions (3′-UTRs) of their target genes (7). When normal bladder epithelia are transformed, as evidenced by alterations in protein expression, dysregulation of miRs is also observed. Previous studies showed that miRs act as either oncogenes or tumor suppressors in bladder cancer. Oncogenic miRs are mainly upregulated (810) and tumor-suppressive miRs are downregulated (1113). Similarly, our previous studies demonstrated that miR-20b-induced p21WAF1-mediated G1 phase cell cycle arrest of bladder cancer EJ cells, suggesting that the miR has a tumor suppressive function (14). In the present study, we reported that miR-892b inhibits proliferation, migration and invasion of bladder cancer EJ cells by regulating the p19ARF/cyclin D1/CDK6 and Sp-1/MMP-9 cascade.

Materials and methods

Materials

Polyclonal antibodies against cyclin E, CDK2, CDK6, cyclin D1, p53, p19ARF, p21WAF1, p27KIP1 and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-MMP-9 polyclonal antibody was obtained from Chemicon International (Billerica, MA, USA). miR-892b (5′-CACUGGCUCCUUUCUGGGUAGA-3′) and miR-892b inhibitor were designed and synthesized by Genolution Pharmaceuticals, Inc. (Seoul, Korea).

Cell cultures

Human bladder carcinoma cell lines (EJ, 5637 and T24) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with glucose supplemented with 10% fetal calf serum, L-glutamine and antibiotics (Biological Industries, Beit Haemek, Israel) at 37°C in a 5% CO2 humidified incubator. Normal human urothelial cells (HUCs) were purchase from ScienCell Research Laboratories (Carlsbad, CA, USA). The cells were maintained in urothelial cell medium with supplements according to the manufacturer's protocol.

Quantitative real-time RT-PCR (qRT-PCR)

Quantification of miRNA expression was performed using a Rotor-Gene™ 6000 as previously described (15). Real-time PCR assays were carried out using the miScript PCR Starter kit (Qiagen Korea, Seoul, Korea). The PCR reaction was performed in a final volume of 20 µl (10 µl 2X QuantiTect SYBR-Green PCR Master Mix, 2 µl 10X miScript Universal Primer (U6), 2 µl 10 pmol forward primer, 2 µl template cDNA and RNase-free water). Real-time PCR conditions were as follows: 1 cycle of initial activation for 15 min at 95°C, followed by 50 cycles of 15 sec at 94°C for denaturation, annealing for 30 sec at 55°C and extension for 30 sec at 70°C. The melting program was performed at 70–99°C at a heating rate of 1°C/5 sec. Spectral data were captured and determined using Rotor-Gene™ Real-Time Analysis Software 6.0, Build 14. All experiments were carried out in triplicate. U6 was used as a control to normalize the quantity of miRNA.

Bioinformatics analysis

Screening for possible targets of miR-892b was performed with the miRanda algorithm (http://www.microrna.org/microrna/home.do) and the NCBI mRNA database (NCBI mRNA DB: http://www.ncbi.nlm.nih.gov/).

Cell proliferation

Cell proliferation was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (16). Cell morphology was photographed using phase-contrast microscopy.

Transfection

Cells were transfected with miR-892b and the miR-892b inhibitor using Lipofectamine 2000 transfection reagent (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer's protocol.

Flow cytometric cell cycle analysis

Cells were harvested and fixed in 70% ethanol. After being washed with ice-cold phosphate-buffered saline (PBS), cells were incubated with RNase (1 mg/ml) followed by propidium iodide (50 mg/ml). The phase distribution of the cell cycle was determined by a Becton-Dickinson FACStar flow cytometer equipped with Becton-Dickinson Cell Fit software.

Immunoblot analysis

Preparation and quantitation of protein lysates were performed as previously described (14). Lysates were electrophoresed on a 10% polyacrylamide gel (SDS-PAGE) under denaturing conditions and transferred to nitrocellulose membranes (Hybond; Amersham Corp., Arlington Heights, IL, USA). The blots were blocked with 5% (wt/vol) non-fat dry milk in Tris-buffered saline (TBS) [10 mM Tris-HCl (pH 8.0), 150 mM NaCl], and then, the membranes were incubated with primary antibodies at 4°C overnight. The blots were then incubated with peroxidase-conjugated secondary antibodies for 90 min. A chemiluminescence reagent kit (Amersham Corp.) was utilized for the detection of the western blot analyses. The experiments were repeated at least 3 times.

Immunoprecipitation and immune complex kinase assays

Cell lysates were prepared using ice-cold lysis buffer as previously described (14). Briefly, after centrifugation of the lysates at 10,000 × g for 5 min, the supernatants were precipitated by protein A sepharose beads pre-coated with the indicated antibodies at 4°C for 2 h. The immunoprecipitated proteins on the beads were washed 4 times with 1 ml of lysis buffer and twice with a kinase buffer (14). Finally, pellets were re-suspended in 25 µl of kinase buffer containing 1 µg glutathione S-transferase (GST)-pRb C-terminal (pRb amino acids 769–921) fusion protein (Santa Cruz Biotechnology), 20 µM/l ATP and 5 µCi of [γ32P]-ATP (4,500 µCi/mmol; ICN). Subsequently, re-suspended pellets were incubated at 30°C for 20 min with occasional mixing. The kinase reactions were terminated by the addition of 25 µl of 2X Laemmli sample buffer and were heated at 100°C for 5 min. Samples were resolved on 10% SDS-polyacrylamide gels, which were then dried. Radioactive bands were visualized. The migration of GST-pRb was determined using Coomassie blue staining.

Wound-healing migration assay

Cells (3×105) were seeded into 6-well plates in 2 ml medium and were grown to 90% confluency. A clear area was created with a 2 mm pipette tip. After 3 washes with PBS, the plates were incubated at 37°C in serum-free medium. Migration of cells into the clear area was analyzed and photographed using an inverted microscope (magnification, ×40).

Invasion assay

Invasion assays were performed using an invasion assay kit (Cell Biolabs, Inc., San Diego, CA, USA) according to the manufacturer's instructions. Cells (2.5×104) were re-suspended in serum-free medium and plated in the upper chamber. Media with 10% FBS was added to the lower chamber as a chemoattractant. After 24 h of incubation, cells in the lower chamber were stained and photographed. Cell invasion was evaluated using a commercial cell invasion assay kit (Chemicon International).

Gelatin zymography

Culture supernatants were resolved on a polyacrylamide gel containing 1 mg/ml gelatin. Gels were washed with 2.5% Triton X-100 at room temperature for 2 h followed by incubation at 37°C overnight in a buffer containing 10 mM CaCl2, 150 mM NaCl and 50 mM Tris-HCl, pH 7.5. The gel was stained with 0.2% Coomassie blue and photographed with a light box. Areas of gelatinase activity were visible as clear bands in a dark blue field.

Preparation of nuclear extracts and EMSA

Nuclear proteins were extracted as previously described (17). Briefly, cells were washed, scraped and suspended in a buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)]. The cells were lysed with 0.5% NP40. The homogenates were then centrifuged, and the nuclear pellets were extracted with an ice-cold high-salt buffer (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF). After centrifugation, the supernatants containing the nuclear extracts were prepared. Protein concentrations were measured using Bradford reagent (Bio-Rad). An electrophoretic mobility shift assay (EMSA) was performed as previously described (17). In brief, the oligonucleotides spanning the MMP-9 cis element of interest were end-labeled with 32P-ATP by T4 polynucleotide kinase (Promega, Madison, WI, USA). Nuclear extracts (10–20 µg) were incubated with a radio-labeled oligonucleotide probe (10,000 cpm) at 4°C for 20 min in a binding buffer solution (25 mM HEPES buffer pH 7.9, 0.5 mM EDTA, 0.5 mM DTT, 0.05 M NaCl, and 2.5% glycerol, and 2 µg poly dI/dC). The DNA-protein complex was resolved at 4°C on a 6% polyacrylamide gel containing a TBE running buffer (89 mM Tris, 89 mM boric acid and 1 mM EDTA). The gel was rinsed, dried and then exposed to X-ray film for 10 h. The sequences for the oligonucleotides were as follows: AP-1, CTGACCCCTGAGTCAGCACTT; NF-κB, CAGTGGAATTCCCCAGCC; and Sp-1, GCCCATTCCTTCCGCCCCCAGATGAAGCAG.

Statistical analysis

Where appropriate, data are expressed as mean ± SE. Data were evaluated by factorial ANOVA and a Fisher's least significant difference test where appropriate. Statistical significance was set at P<0.05.

Results

Basal level of miR-892b expression is low in bladder cancer cells

The aim of the present study was to verify the role of miR-892b in bladder cancer. We first measured the basal expression level of miR-892b in 3 different bladder cancer cell lines, EJ, T-24 and 5637, and one normal human urothelial cell line (HUC). Quantitative real-time-PCR showed that the basal levels of miR-892b were significantly lower in all the 3 bladder cancer cells than in HUC cells (Fig. 1A). EJ and T24 cells exhibited the least expression of the 3 cancer cells; therefore, we selected EJ and T24 cells for further experiments. We then investigated the effect of miR-892b on the proliferation of EJ and T24 cells, and the cells were transfected with Lipofectamine 2000 (control), miR-892b mimic (miR-892b), and inhibitor of miR-892b (inhibitor) at concentrations from 10 to 100 nM. Introducing miR-892b mimic into EJ cells significantly inhibited proliferation in a dose-dependent manner (Fig. 1B). In contrast, neither control nor inhibitor showed any meaningful decrease in the growth of the cells. Images of the transfected cells supported the result (Fig. 1C). Similar results were obtained from T24 cells (Fig. 1B and C). Subsequent experiments were performed with a miR-892b concentration of 30 nM (~IC50) for EJ cells and 50 nM (~IC50) for T24 cells, respectively.

miR-892b causes G1 phase cell cycle arrest in bladder cancer cells

To elucidate the cause of miR-892b-mediated growth inhibition of bladder cancer cells, we investigated the cell cycle phase distribution of miR-892b-transfected cells through flow cytometric analysis. As demonstrated by the DNA histograms of the cell cycle, introduction of miR-892b resulted in accumulation in the G1 phase (Fig. 2A–D). In agreement with that result, G1 phase accumulation was followed by a corresponding decrease in the S phase population (Fig. 2A–D). Based on the results of flow cytometry, we searched the microRNA database (miRanda) to find possible targets of miR-892b in terms of cell cycle regulators, including cyclins, CDKs and CDK inhibitors. We identified cyclin D1, CDK6 and p19ARF as possible causes of G1 phase cell cycle arrest in bladder cancer EJ cells. First, we investigated changes in protein expression levels of the G1 phase cell cycle regulators in miR-892b transfectants using immunoblots. In line with bioinformatics analysis, the expression of cyclin D1 and CDK6 were significantly reduced in miR-892b transfectants (Fig. 3A). However, the expression levels of cyclin E and CDK2 were not significantly altered. Progression of the cell cycle often requires activation of CDKs, which form a complex with corresponding cyclins. CDK6 couples with cyclin D1 and phosphorylates Rb (18). We investigated the kinase activity of CDK6 in miR-892b transfectants with immunoprecipitation assays. In accordance with the immunoblot results (Fig. 3A), the kinase activity of CDK6 was reduced by ~50% in miR-892b transfectants compared to the control or the miR-892b inhibitor (Fig. 3C). This strongly suggests that miR-892b targets cyclin D1-associated growth signals in bladder cancer cells.

miR-892b leads to the upregulation of p19ARF, resulting in growth inhibition of bladder cancer cells

Based on the finding that miR-892b-induced G1 phase cell cycle arrest in bladder cancer EJ cells, we investigated the effect of miR-892b on protein levels of CDK inhibitors associated with G1 to S phase cell cycle progression. Immunoblot analysis demonstrated upregulation of p19ARF in miR-892b-transfected EJ cells (Fig. 3B). However, expression levels of p21WAF1/CIP1, p27KIP1 and p53 were unchanged (Fig. 3B). Upregulation of p19ARF by miR-892b was confirmed via immunoprecipitation experiments with CDK6 followed by immunoblotting with p19ARF or CDK6 (control). p19ARF/CDK6 complex formation was increased by ~50% in miR-20b-transfected cells compared with control cells (Fig. 3D). Taken together, these results demonstrate that p19ARF is a critical effector in miR-892b-mediated G1 phase cell cycle arrest of bladder cancer EJ cells.

miR-892b reduces wound-healing migration and invasiveness of bladder cancer cells

During the progression of bladder cancer, transformed cells frequently acquire the ability to invade other tissues. We investigated the effect of miR-892b on the migration and invasion of bladder cancer cells through wound-healing and Boyden chamber invasion assays. As shown in Fig. 4A, miR-892b transfectants exhibited slower recovery in a wounded area than did control or miR-892b inhibitor-tranfected cells. Wound-closure rate was ~60% lower in the miR-892b transfectants than in the control. Similarly, Boyden chamber invasion assays showed that invasiveness into the Matrigel-coated chamber was reduced >50% compared to the control or the inhibitor (Fig. 4B). Taken together, these results suggest that miR-892b has tumor-suppressive effects that regulate cellular motility via migration and invasion.

miR-892b downregulates MMP-9 expression via activation of the transcription factor SP-1

In order to identify the molecular targets responsible for the inhibition of invasion and migration caused by miR-892b, we used miRanda to search for microRNA targets and found MMP-9 as a candidate (Fig. 5C). Since MMP-2, -3, -7 and -9 were previously reported to be key molecules associated with aggressiveness and poor prognosis in bladder cancer (19,20), we examined the protein expression levels of MMP-2 and MMP-9 in miR-892b-transfected cells using gelatin zymography assays. As shown in Fig. 5A, expression of MMP-9 was significantly reduced in the growth medium obtained from miR-892b-transfected cells. However, no significant changes in MMP-9 levels were observed in the medium from the control or the miR-892b inhibitor cells. Immunoblot analysis of the miR-892b transfectants also showed a reduction in MMP-9 expression (Fig. 5A). miRanda bioinformatics analysis for microRNA targets identified Sp-1 as one of the targets of miR-892b. Thus, using EMSA assays, we investigated whether transcription factors including Sp-1, AP-1 and NF-κB are modulated by miR-892b transfection in EJ cancer cells. Nuclear extracts from miR-892b transfectants exhibited a reduction in activity of the Sp-1 binding motif (Fig. 5B). However, no specific binding activity of AP-1 or NF-κB was observed in the transfectants (Fig. 5B). Taken together, these results suggest that the Sp-1 transcription factor is involved in the suppression of MMP-9-mediated migration and invasion of bladder cancer EJ cells induced by miR-892b.

Discussion

Bladder cancer is one of the most common types of cancers worldwide, causing annually ~150,000 deaths (21). Standard therapeutic options to date for advanced bladder cancer consist of cisplatin-based combinatorial chemotherapies, although new therapeutic approaches are being explored. Earlier studies by others demonstrated that microRNAs regulate cellular functions such as cellular proliferation, differentiation and cell death (7,22). miR-892b was previously found to be a modulator of MCL1 in colorectal cancer, sensitizing cells to a BCL inhibitor, ABT-263 (navitoclax) (23). However, the cellular function of miR-892b and its cellular signaling networks in cancer states have not been reported.

Based on predicted-target bioinformatics analysis of miR-892b, we found that miR-892b regulates signaling molecules associated with cellular processes including proliferation, migration and invasion of bladder cancer cells. Upon initial evaluation, the basal expression of miR-892b was shown to be significantly lower in all bladder cancer cell lines tested than in normal human urothelial cells, which indicated that miR-892b may be associated with transformation of bladder cells. As speculated, transfection of a miR-892b mimic to bladder cancer cells resulted in growth arrest. Transfection of a miR-892b inhibitor did not cause any alteration in the growth of bladder cancer cells. These results suggest that, in bladder cancers, a cellular mechanism exists to downregulate miR-892b, which limits its tumor suppressive function. In addition, we found that miR-892b participates in the progression of the cell cycle from G1 to S phase in bladder cancer cells. Among known G1 phase cell cycle regulators, we examined the expression level of cyclin D1, cyclin E, CDK2 and CDK6. Cyclin D and E are key regulators of the cell cycle and are associated with the initiation and progression of bladder cancer (24,25). We found that cyclin D1, but not cyclin E, was downregulated in miR-892b-transfected EJ bladder cancer cells. Further immune complex kinase assays showed that the activity of CDK6, but not that of CDK2, was reduced by miR-892b transfection. Notably, Zhao et al reported that targeting CDK6 in bladder cancer cells by transfecting miR-29c reduced the growth and invasiveness of bladder cancer cells (26). In agreement with that result, we found that introduction of miR-892b to bladder cancer cells significantly inhibited proliferation by lowering protein levels of cyclin D1 and CDK6. The activity of CDKs is tightly controlled by CDK inhibitors such as p16INK4A, p19ARF, p21WAF1/CIP1 and p27KIP1. Mutations in CDKs are frequently observed in certain types of cancer, as in the case of p16INK4A in melanoma (27). However, signature mutations or mutation hot spots in CDKs for bladder cancers are relatively understudied. Previously, the correlation of p21WAF1/CIP1, p27KIP1 and p53 levels were examined in 51 patient-derived tumor specimens with overall survival rate. They concluded that downregulation of p27KIP1 and overexpression of cyclin D1 and D3 predicted survival of bladder cancer patients (28). Another recent study showed that miR-451 inhibits the proliferation of esophageal carcinoma cells by targeting p19ARF (29). In the present study, p19ARF was upregulated in miR-892b transfectants. However, p21WAF1/CIP1 and p27KIP1 levels remained unchanged. These results demonstrated for the first time that miR-892b impedes proliferation of bladder cancer cells through G1 phase cell cycle arrest by inducing the p19ARF/cyclin D1/CDK6 cascade pathway.

Managing invasiveness of bladder cancer is one of the most difficult clinical challenges. The 5-year survival rate for patients with stage 4 bladder cancer is ~10% for both men and women, which is attributable to invasiveness or metastasis (1). In the present study, we found that miR-892b inhibits the invasion and migration of bladder cancer cells through wound recovery and Boyden chamber invasion assays. During disease progression, bladder cancer cells penetrate surrounding muscle tissues through the generation of proteolytic enzymes such as matrix methalloproteinases (MMPs) (30). Previous studies demonstrated that MMPs are closely correlated with poor prognosis in bladder cancer patients (19,20,31). Similarly, we previously identified that MMP-9, but not MMP-2, was associated with invasion of bladder cancer cells (14,17,33). The activity of MMP-9 in bladder cancer cells is tightly regulated by transcription factors, including Sp-1, AP-1 and NF-κB (17,32,33). In our bioinformatic analysis, Sp-1 was identified as a binding candidate for miR-892b. Utilizing EMSA assays, we verified that Sp-1 was the critical transcription factor regulated by miR-892b. However, neither AP-1 nor NF-κB was regulated by mir-892b. Our data suggest that Sp-1 is essential for miR-892b-mediated suppression of MMP-9 expression leading to the reduction in the migration and invasion of bladder cancer cells.

In conclusion, we demonstrated that aberrant expression of miR-892b is associated with progression of bladder cancer cells. Re-introduction of a miR-892b mimic significantly inhibits the proliferation and migration of bladder cancer cells, which suggests that miR-892b may be a novel target for treatment of bladder cancer.

Acknowledgments

The present study was supported by the National Research Foundation of Korea (NRF) grant by the government of Korea (MSIP) (no. 2014007036). This study was also supported by the Functional Districts of the Science Belt support program, Ministry of Science, ICT and Future Planning (2015K000284).

References

1 

American Cancer Society: Cancer Facts & Figures 2015. American Cancer Society, Inc.; Atlanta, GA: 2015

2 

Kaufman DS, Shipley WU and Feldman AS: Bladder cancer. Lancet. 374:239–249. 2009. View Article : Google Scholar : PubMed/NCBI

3 

Lindgren D, Liedberg F, Andersson A, Chebil G, Gudjonsson S, Borg A, Månsson W, Fioretos T and Höglund M: Molecular characterization of early-stage bladder carcinomas by expression profiles, FGFR3 mutation status, and loss of 9q. Oncogene. 25:2685–2696. 2006. View Article : Google Scholar : PubMed/NCBI

4 

Habuchi T, Marberger M, Droller MJ, Hemstreet GP III, Grossman HB, Schalken JA, Schmitz-Dräger BJ, Murphy WM, Bono AV, Goebell P, et al: Prognostic markers for bladder cancer: International Consensus Panel on bladder tumor markers. Urology. 66(Suppl 1): S64–S74. 2005. View Article : Google Scholar

5 

Kamai T, Takagi K, Asami H, Ito Y, Oshima H and Yoshida KI: Decreasing of p27Kip1 and cyclin E protein levels is associated with progression from superficial into invasive bladder cancer. Br J Cancer. 84:1242–1251. 2001. View Article : Google Scholar : PubMed/NCBI

6 

Liukkonen T, Rajala P, Raitanen M, Rintala E, Kaasinen E and Lipponen P; The Finnbladder Group: Prognostic value of MIB-1 score, p53, EGFr, mitotic index and papillary status in primary superficial (Stage pTa/T1) bladder cancer: A prospective comparative study. Eur Urol. 36:393–400. 1999. View Article : Google Scholar : PubMed/NCBI

7 

Bartel DP: MicroRNAs: Target recognition and regulatory functions. Cell. 136:215–233. 2009. View Article : Google Scholar : PubMed/NCBI

8 

Wu Z, Liu K, Wang Y, Xu Z, Meng J and Gu S: Upregulation of microRNA-96 and its oncogenic functions by targeting CDKN1A in bladder cancer. Cancer Cell Int. 15:1072015. View Article : Google Scholar : PubMed/NCBI

9 

Lei M, Xie W, Sun E, Sun Y, Tian D, Liu C, Han R, Li N, Liu M, Han R, et al: microRNA-21 regulates cell proliferation and migration and cross talk with PTEN and p53 in bladder cancer. DNA Cell Biol. 34:626–632. 2015. View Article : Google Scholar : PubMed/NCBI

10 

Xiu Y, Liu Z, Xia S, Jin C, Yin H, Zhao W and Wu Q: MicroRNA-137 upregulation increases bladder cancer cell proliferation and invasion by targeting PAQR3. PLoS One. 9:e1097342014. View Article : Google Scholar : PubMed/NCBI

11 

Feng Y, Kang Y, He Y, Liu J, Liang B, Yang P and Yu Z: microRNA-99a acts as a tumor suppressor and is down-regulated in bladder cancer. BMC Urol. 14:502014. View Article : Google Scholar : PubMed/NCBI

12 

Wu D, Zhou Y, Pan H, Zhou J, Fan Y and Qu P: microRNA-99a inhibiting cell proliferation, migration and invasion by targeting fibroblast growth factor receptor 3 in bladder cancer. Oncol Lett. 7:1219–1224. 2014.PubMed/NCBI

13 

Zhang J, Wang S, Han F, Li J, Yu L, Zhou P, Chen Z, Xue S, Dai C and Li Q: MicroRNA-542-3p suppresses cellular proliferation of bladder cancer cells through post-transcriptionally regulating survivin. Gene. 579:146–152. 2016. View Article : Google Scholar : PubMed/NCBI

14 

Park SL, Cho TM, Won SY, Song JH, Noh DH, Kim WJ and Moon SK: MicroRNA-20b inhibits the proliferation, migration and invasion of bladder cancer EJ cells via the targeting of cell cycle regulation and Sp-1-mediated MMP-2 expression. Oncol Rep. 34:1605–1612. 2015.PubMed/NCBI

15 

Yun SJ, Jeong P, Kim WT, Kim TH, Lee YS, Song PH, Choi YH, Kim IY, Moon SK and Kim WJ: Cell-free microRNAs in urine as diagnostic and prognostic biomarkers of bladder cancer. Int J Oncol. 41:1871–1878. 2012.PubMed/NCBI

16 

Moon SK, Jung SY, Choi YH, Lee YC, Patterson C and Kim CH: PDTC, metal chelating compound, induces G1 phase cell cycle arrest in vascular smooth muscle cells through inducing p21Cip1 expression: Involvement of p38 mitogen activated protein kinase. J Cell Physiol. 198:310–323. 2004. View Article : Google Scholar

17 

Lee SJ, Cho SC, Lee EJ, Kim S, Lee SB, Lim JH, Choi YH, Kim WJ and Moon SK: Interleukin-20 promotes migration of bladder cancer cells through extracellular signal-regulated kinase (ERK)-mediated MMP-9 protein expression leading to nuclear factor (NF-κB) activation by inducing the up-regulation of p21WAF1 protein expression. J Biol Chem. 288:5539–5552. 2013. View Article : Google Scholar

18 

Sherr CJ and Roberts JM: CDK inhibitors: Positive and negative regulators of G1-phase progression. Genes Dev. 13:1501–1512. 1999. View Article : Google Scholar : PubMed/NCBI

19 

Reis ST, Leite KR, Piovesan LF, Pontes-Junior J, Viana NI, Abe DK, Crippa A, Moura CM, Adonias SP, Srougi M, et al: Increased expression of MMP-9 and IL-8 are correlated with poor prognosis of Bladder Cancer. BMC Urol. 12:182012. View Article : Google Scholar : PubMed/NCBI

20 

Seiler R, Thalmann GN and Fleischmann A: MMP-2 and MMP-9 in lymph-node-positive bladder cancer. J Clin Pathol. 64:1078–1082. 2011. View Article : Google Scholar : PubMed/NCBI

21 

Siegel R, Naishadham D and Jemal A: Cancer statistics, 2013. CA Cancer J Clin. 63:11–30. 2013. View Article : Google Scholar : PubMed/NCBI

22 

Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP and Bartel DP: MicroRNA targeting specificity in mammals: Determinants beyond seed pairing. Mol Cell. 27:91–105. 2007. View Article : Google Scholar : PubMed/NCBI

23 

Lam LT, Lu X, Zhang H, Lesniewski R, Rosenberg S and Semizarov D: A microRNA screen to identify modulators of sensitivity to BCL2 inhibitor ABT-263 (navitoclax). Mol Cancer Ther. 9:2943–2950. 2010. View Article : Google Scholar : PubMed/NCBI

24 

Eissa S, Ahmed MI, Said H, Zaghlool A and El-Ahmady O: Cell cycle regulators in bladder cancer: Relationship to schistosomiasis. IUBMB Life. 56:557–564. 2004. View Article : Google Scholar : PubMed/NCBI

25 

Shan G and Tang T: Expression of cyclin D1 and cyclin E in urothelial bladder carcinoma detected in tissue chips using a quantum dot immunofluorescence technique. Oncol Lett. 10:1271–1276. 2015.PubMed/NCBI

26 

Zhao X, Li J, Huang S, Wan X, Luo H and Wu D: MiRNA-29c regulates cell growth and invasion by targeting CDK6 in bladder cancer. Am J Transl Res. 7:1382–1389. 2015.PubMed/NCBI

27 

FitzGerald MG, Harkin DP, Silva-Arrieta S, MacDonald DJ, Lucchina LC, Unsal H, O'Neill E, Koh J, Finkelstein DM, Isselbacher KJ, et al: Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma: Analysis of a clinic-based population. Proc Natl Acad Sci USA. 93:8541–8545. 1996. View Article : Google Scholar : PubMed/NCBI

28 

Lopez-Beltran A, Luque RJ, Alvarez-Kindelan J, Quintero A, Merlo F, Carrasco JC, Requena MJ and Montironi R: Prognostic factors in stage T1 grade 3 bladder cancer survival: The role of G1-S modulators (p53, p21Waf1, p27kip1, Cyclin D1, and Cyclin D3) and proliferation index (ki67-MIB1). Eur Urol. 45:606–612. 2004. View Article : Google Scholar : PubMed/NCBI

29 

Zang WQ, Yang X, Wang T, Wang YY, Du YW, Chen XN, Li M and Zhao GQ: MiR-451 inhibits proliferation of esophageal carcinoma cell line EC9706 by targeting CDKN2D and MAP3K1. World J Gastroenterol. 21:5867–5876. 2015.PubMed/NCBI

30 

Roy R, Yang J and Moses MA: Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer. J Clin Oncol. 27:5287–5297. 2009. View Article : Google Scholar : PubMed/NCBI

31 

Ramón de Fata F, Ferruelo A, Andrés G, Gimbernat H, Sánchez-Chapado M and Angulo JC: The role of matrix metalloproteinase MMP-9 and TIMP-2 tissue inhibitor of metalloproteinases as serum markers of bladder cancer. Actas Urol Esp. 37:480–488. 2013. View Article : Google Scholar : PubMed/NCBI

32 

Mian BM, Dinney CP, Bermejo CE, Sweeney P, Tellez C, Yang XD, Gudas JM, McConkey DJ and Bar-Eli M: Fully human anti-interleukin 8 antibody inhibits tumor growth in orthotopic bladder cancer xenografts via down-regulation of matrix metal-loproteases and nuclear factor-kappaB. Clin Cancer Res. 9:3167–3175. 2003.PubMed/NCBI

33 

Lee EJ, Lee SJ, Kim S, Cho SC, Choi YH, Kim WJ and Moon SK: Interleukin-5 enhances the migration and invasion of bladder cancer cells via ERK1/2-mediated MMP-9/NF-κB/AP-1 pathway: Involvement of the p21WAF1 expression. Cell Signal. 25:2025–2038. 2013. View Article : Google Scholar : PubMed/NCBI

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October-2016
Volume 36 Issue 4

Print ISSN: 1021-335X
Online ISSN:1791-2431

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Copy and paste a formatted citation
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Spandidos Publications style
Shin S, Park S, Hwang B, Moon B, Kim WT, Kim W and Moon S: MicroRNA-892b influences proliferation, migration and invasion of bladder cancer cells by mediating the p19ARF/cyclin D1/CDK6 and Sp-1/MMP-9 pathways. Oncol Rep 36: 2313-2320, 2016
APA
Shin, S., Park, S., Hwang, B., Moon, B., Kim, W.T., Kim, W., & Moon, S. (2016). MicroRNA-892b influences proliferation, migration and invasion of bladder cancer cells by mediating the p19ARF/cyclin D1/CDK6 and Sp-1/MMP-9 pathways. Oncology Reports, 36, 2313-2320. https://doi.org/10.3892/or.2016.5052
MLA
Shin, S., Park, S., Hwang, B., Moon, B., Kim, W. T., Kim, W., Moon, S."MicroRNA-892b influences proliferation, migration and invasion of bladder cancer cells by mediating the p19ARF/cyclin D1/CDK6 and Sp-1/MMP-9 pathways". Oncology Reports 36.4 (2016): 2313-2320.
Chicago
Shin, S., Park, S., Hwang, B., Moon, B., Kim, W. T., Kim, W., Moon, S."MicroRNA-892b influences proliferation, migration and invasion of bladder cancer cells by mediating the p19ARF/cyclin D1/CDK6 and Sp-1/MMP-9 pathways". Oncology Reports 36, no. 4 (2016): 2313-2320. https://doi.org/10.3892/or.2016.5052