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Introduction

Bladder cancer is the most frequent malignancy of the urinary tract and the seventh most common cancer worldwide (1,2). There are ~429,800 new cases and 165,100 mortalities associated with bladder cancer every year worldwide (3). The morbidity of bladder cancer is higher in developed countries than in developing countries, due to the aging population, and the increase in occupational chemical exposure and the use of cigarettes (4,5). Despite advancements in current therapeutic treatments, the clinical outcome of patients with bladder cancer remains poor. The 5-year overall survival rate for patients with non-muscle invasive bladder cancer is ~90%, however, it is ~60% for patients with muscle-invasive bladder cancer (6,7). A major issue associated with bladder cancer therapy is that following conventional treatments including surgical resection, chemotherapy and radiotherapy, a large proportion of patients suffer recurrence and metastasis, however, there have been a few advances in clinical practice (8). Therefore, it is important to elucidate the mechanisms underlying bladder cancer initiation and progression, as they may provide novel therapeutic targets for the treatments of bladder cancer.

MicroRNAs (miRNAs or miRs) are a large class of small, endogenous, non-coding and single-stranded RNA molecules of ~22 nucleotides (9). Based on miRBase version 21, released in June 2014 (http://www.mirbase.org/), 1,881 miRNA precursors and 2,588 mature miRNAs have been identified in the human genome. Mature miRNAs negatively regulate gene expression through imperfect complementary sequence pairing to the 3′-untranslated regions (3′UTRs) of their target genes, subsequently promoting translational inhibition or mRNA degradation, which results in moderate protein expression levels (9,10). Functionally, miRNAs are implicated in a range of biological processes, including cell proliferation and cycling, apoptosis, differentiation, generation and metastasis (11). In addition, abnormal expression of miRNAs has been frequently observed in a number of different types of human cancer, including bladder (12), prostate (13), gastric (14) and colorectal cancers (15). Previous studies have demonstrated that miRNAs can act as tumor suppressors or oncogenes in human cancers, and are therefore potential therapeutic targets for cancer diagnosis, treatment and prognosis (16–18).

miR-539 has been studied in multiple different types of human cancers (19–21). However, there is currently no information available concerning miR-539 in bladder cancer. In the present study, the expression levels of miR-539 were determined in bladder cancer tissues and cell lines. In vitro functional assays were performed to investigate the effects of miR-539 on bladder cancer cell proliferation and invasion. In addition, the molecular mechanism underlying the effects of miR-539 on cell proliferation and invasion was also evaluated.

Materials and methods

Clinical specimens

The present study was approved by the Medical Ethics Committee of the Affiliated Hospital of Guizhou Medical University (Guizhou, China). Informed written consent was also obtained from all subjects. All experimental protocols were carried out in accordance with the approved guidelines (22). Bladder cancer tissues (n=49) and matched adjacent normal bladder tissues (n=49) were obtained from patients (n=49; age range, 46–78 years; Table I) who underwent surgery in the Affiliated Hospital of Guizhou Medical University from May 2012 to March 2014. All tissues were freshly frozen in liquid nitrogen and stored at −80°C until required.

Table I. Associations between miR-539 expression and clinicopathological features in patients with bladder cancer.

Table I.

Associations between miR-539 expression and clinicopathological features in patients with bladder cancer.

		miR-539 expression
Clinicopathological feature	Number of cases (n)	Low (n)	High (n)	P-value
Sex				0.079
Male	33	16	17
Female	16	12	4
Age (years)				0.620
<60	23	14	9
≥60	26	14	12
Tumor number				0.665
Single	32	19	13
Multiple	17	9	8
Tumor grade				0.804
I–II	22	13	9
III	27	15	12
Tumor stage				0.013a
T1-T2	25	10	15
T3-T4	24	18	6
Lymph node metastasis				0.017a
Positive	26	19	7
Negative	23	9	14

{ label (or @symbol) needed for fn[@id='tfn1-mmr-17-04-4917'] } Clinicopathological features were analyzed and statistically compared with the level of miR-539 expression to determine their associations in patients with bladder cancer (n=49).

a P<0.05. miR, microRNA.

Cell lines and cell culture

Bladder cancer cell lines (T24, 5637 and TCCSUP) and the normal bladder epithelial cell line (SV-HUC-1) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). All cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin G and 100 mg/ml streptomycin in a 5% CO2 incubator at 37°C. Cell passage was performed once the cell density had reached 90%.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay

Total RNA was isolated from all tissues and cells (1×106) using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The One Step SYBR® PrimeScript™ miRNA RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) was used to analyze the levels of miR-539 expression, according to the manufacturer's instructions. The thermocycling conditions were as follows: 42°C for 5 min, 95°C for 10 sec, followed by 40 cycles of 95°C for 5 sec, 55°C for 30 sec and 72°C for 30 sec. To quantify insulin like growth factor 1 receptor (IGF-1R) mRNA expression, reverse transcription was performed using the Moloney Murine Leukemia Virus Reverse Transcription system (Promega Corporation, Madison, WI, USA), followed by qPCR using the SYBR Green I mix (Takara Biotechnology Co., Ltd.), according to the manufacturer's instructions. The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. U6 and GADPH were used as reference genes for miR-539 and IGF-1R, respectively. The following primers were used: miR-539, forward, 5′-GAAGAGGCTAACGTGAGGTTG-3′ and reverse, 5′-CACCATGACCAAGCCACGTAG-3′; U6, forward, 5′-CTCGCTTCGGCAGCACATATACT-3′ and reverse, 5′-ACGCTTCACGAATTTGCGTGTC-3′; IGF-1R, forward, 5′-GGCATACCTCAACGCCAATA-3′ and reverse, 5′-CAGCCCTTTCCCTCCTTT-3′; GAPDH, forward, 5′-ATAGCACAGCCTGGATAGCAACGTAC-3′ and reverse, 5′-CACCTTCTACAATGAGCTGCGTGTG-3′. Each sample was analyzed in triplicate and experiments were repeated three times. The relative expression was analyzed using the 2−ΔΔCq method (23).

Oligonucleotide transfection

miR-539 mimics, the corresponding negative control mimics (miR-NC), small interfering RNA of IGF-1R (si-IGF-1R) and the corresponding negative controls (si-NC) were obtained from Shanghai GenePharma Co., Ltd., Shanghai, China. The sequences were as follows: miR-539 mimics, 5′-GGAGAAAUUAUCCUUGGUGUGU-3′; miR-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′; si-IGF-1R, 5′-CAACGGCCTATTGTCAGGT-3′; and si-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′. For functional assays, T24 cells were seeded into 6-well plates at a density of 60–70% confluency. Cells were then transfected with miR-539 mimics (100 pmol), miR-NC (100 pmol), si-IGF-1R (100 pmol) or si-NC (100 pmol) using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc.) following to the manufacturer's instructions. A MTT assay was performed at 24 h post-transfection, and RT-qPCR and cell invasion assays were performed at 48 h post-transfection. Following 72 h after transfection, western blot analysis was used to detect protein expression.

MTT assay

Cell proliferation was determined by performing an MTT assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Briefly, transfected cells were re-seeded in 96-well culture plates at a density of 1×103 cells/well. Cells were then incubated at 37°C for 24, 48, 72 or 96 h. At each time point, 10 µl MTT (5 mg/ml) was added into each well and incubated for an additional 4 h. The solution containing the MTT regent was then carefully removed and replaced with 150 µl DMSO (Sigma-Aldrich; Merck KGaA). The optical density was determined at a wavelength of 490 nm using an enzyme-linked immunosorbent assay reader (Elx800; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell invasion assay

Matrigel (BD Biosciences, San Jose, CA, USA) coated Transwell chambers (8 µm; Costar; Thermo Fisher Scientific, Inc.) were used to perform cell invasion assay. Briefly, 5×104 transfected cells were cultured in DMEM with 2% FBS (Gibco; Thermo Fisher Scientific, Inc.) and re-seeded into the top chambers, while the lower chambers were filled with DMEM containing 20% FBS (Gibco; Thermo Fisher Scientific, Inc.) as a chemoattractant. Following 48 h of incubation at 37°C, cells that had not crossed over the Matrigel were removed carefully using cotton swabs. The invaded cells were fixed with 100% methanol (Beyotime Institute of Biotechnology, Haimen, China) for 10 min at room temperature, stained with 0.5% crystal violet (Beyotime Institute of Biotechnology) for 10 min at room temperature, washed in PBS (Gibco; Thermo Fisher Scientific, Inc.), photographed and then counted using an Olympus fluorescence microscope (Olympus Corporation, Tokyo, Japan). Five visual fields of each membrane were counted for every Transwell chamber (original magnification, ×100).

Bioinformatics analysis

TargetScan (version 7.1; www.targetscan.org/) and miRanda (www.microrna.org/microrna/) were used to analyze the potential targets of miR-539.

Luciferase reporter assay

Luciferase reporter vectors, psiCHECK-IGF-1R-3′UTR wile type (WT) and psiCHECK-IGF-1R-3′UTR mutant (MUT), were synthesized and confirmed by Shanghai GenePharma Co., Ltd. For the luciferase reporter assay, psiCHECK-IGF-1R-3′UTR WT or psiCHECK-IGF-1R-3′UTR MUT were co-transfected with miR-539 mimics or miR-NC into HEK293T cells (1×105 cells/well) (Shanghai Institute of Biochemistry and Cell Biology) using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc.). Following 48 h incubation at 37°C, cells were collected and luciferase activities were determined using the Dual-Luciferase® Reporter Assay system (Promega Corporation) in accordance with the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity. Each assay was performed in triplicate.

Western blot analysis

Total protein was extracted from transfected cells (6-well plates; 1×106 cells/well) using ice-cold radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology). The concentration of total proteins was quantified using a Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal proteins (20 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The PVDF membranes were blocked in 5% skim milk containing Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h at room temperature. Membranes were then incubated overnight at 4°C with the following antibodies: Mouse anti-human monoclonal IGF-1R (cat. no. sc-81464; 1:1,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse anti-human monoclonal extracellular signal-regulated kinases (ERK; cat. no. sc-514302; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), mouse anti-human monoclonal phosphorylated (p)-ERK (cat. no. sc-81492; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), rabbit anti-human polyclonal protein kinase B (AKT; cat. no. sc-8312; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), mouse anti-human monoclonal p-AKT (cat. no. sc-514032; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.) and mouse anti-human monoclonal GADPH (cat. no. sc-166574; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.). Following 3 washes with TBST every 10 min, the membranes were probed with the corresponding horseradish peroxidase-conjugated secondary antibody (cat. nos. sc-2004 for AKT reactions and sc-2005 for the remaining primary antibodies; 1:5,000 dilution; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature and washed again three times with TBST every 10 min. Visualization was performed using an enhanced chemiluminescence solution (Pierce; Thermo Fisher Scientific, Inc.). ImageJ v1.49 (National Institutes of Health, Bethesda, MD, USA) was used to perform densitometry. GADPH was used as an internal control and three experimental repeats were performed.

Statistical analysis

All data are expressed as the mean ± standard deviation. Analysis was performed with SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA). A paired Student's t-test and Pearson's Χ2 test were applied. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-539 is poorly expressed in clinical bladder cancer tissues and cell lines

To explore the biological roles of miR-539 in bladder cancer, miR-539 expression was measured in bladder cancer tissues using RT-qPCR. The results revealed that the expression levels of miR-539 were reduced in bladder cancer tissues when compared with the matched adjacent normal bladder tissues (P<0.05; Fig. 1A). The expression of miR-539 was further determined in the bladder cancer cell lines T24, 5637 and TCCSUP), and the normal bladder epithelial cell line SV-HUC-1. As observed in the collected tissues, miR-539 was significantly downregulated in all 3 bladder cancer cell lines when compared with the SV-HUC-1 cell line (P<0.05; Fig. 1B).

Figure 1. Expression of miR-539 in bladder cancer. (A) miR-539 was expressed in significantly lower levels in bladder cancer tissues than in matched adjacent normal bladder tissues. *P<0.05, vs. matched adjacent normal bladder tissues. (B) miR-539 expression levels in bladder cancer cell lines (T24, 5637 and TCCSUP) were compared with those of the normal bladder epithelial cell line (SV-HUC-1) following reverse transcription-quantitative polymerase chain reaction. *P<0.05 vs. SV-HUC-1 (normal bladder epithelial cell line). miR, microRNA.

Association between miR-539 expression and clinicopathological features of bladder cancer

The associations between miR-539 expression and the clinicopathological features of bladder cancer were then evaluated. Statistical analysis demonstrated that the expression levels of miR-539 were significantly associated with tumor stage (P=0.013) and lymph node metastasis (P=0.017; Table I). However, there were no significant associations with sex, age, tumor number or tumor grade (all P>0.05; Table I).

miR-539 represses cell proliferation and invasion in bladder cancer

To examine the roles of miR-539 in bladder cancer, miR-539 mimics were transfected into T24 cells to increase miR-539 expression (P<0.05; Fig. 2A). The effect of miR-539 overexpression on cell proliferation was investigated using an MTT assay. As demonstrated in Fig. 2B, transfection of miR-539 mimics significantly inhibited T24 cell proliferation following 96 h (P<0.05). The cell invasive capacity was evaluated by performing a cell invasion assay. The results revealed that upregulation of miR-539 decreased the invasive ability of T24 cells (P<0.05; Fig. 2C). These results suggested that miR-539 inhibits cell proliferation and invasion in bladder cancer.

Figure 2. miR-539 inhibits the proliferation and invasion of T24 cells. (A) Expression of miR-539 in T24 cells transfected with miR-539 mimics or miR-NC was measured by reverse transcription-quantitative polymerase chain reaction. (B) An MTT assay was performed to assess proliferation in T24 cells transfected with miR-539 mimics or miR-NC. Transfection with miR-539 mimics significantly inhibited T24 cell proliferation following 96 h. (C) A cell invasion assay was performed to evaluate the invasive capacity of T24 cells transfected with miR-539 mimics or miR-NC (magnification, ×200). *P<0.05 vs. miR-NC. miR, microRNA; NC, negative control.

IGF-1R is a direct target of miR-539 in bladder cancer

Bioinformatics analysis was used to predict the potential targets of miR-539. Among these candidate target genes, the 3′UTR of the IGF-1R gene contains a putative region that matches the seed sequence of miR-539 (Fig. 3A). IGF-1R was subsequently chosen for further analysis as it has previously been reported to be upregulated in bladder cancer (24) and be involved in tumorigenesis and the progression of bladder cancer (25–28). To confirm our hypothesis, a luciferase reporter assay was performed in HEK293T cells co-transfected with miR-539 mimics or miR-NC, and psiCHECK-IGF-1R-3′UTR WT or psiCHECK-IGF-1R-3′UTR MUT. As shown in Fig. 3B, miR-539 expression significantly decreased luciferase activities in the vector with the wild-type construct (P<0.05), however, not in the mutant IGF-1R 3′UTR construct. IGF-1R mRNA and protein expression were then assessed in T24 cells transfected with miR-539 mimics or miR-NC. As presented in Fig. 3C and D, miR-539 mimic transfection significantly suppressed IGF-1R mRNA and protein expression (P<0.05). These findings indicated that IGF-1R may be a direct target gene of miR-539.

Figure 3. IGF-1R is a direct target gene of miR-539. (A) The putative seed-matching sites and mutant sites between miR-539 and the 3′UTR of the IGF-1R gene. (B) Luciferase activities were determined in HEK293T cells co-transfected with WT or MUT reporter, and miR-539 mimics or miR-NC. (C) T24 cells were transfected with miR-539 mimics or miR-NC. Following 48 h of transfection, reverse transcription-quantitative polymerase chain reaction was performed to detect IGF-1R mRNA expression. (D) Total proteins were collected from T24 cells transfected with miR-539 mimics or miR-NC, and subjected to western blot analysis for IGF-1R protein expression. The results were normalized to those of GAPDH. *P<0.05 vs. miR-NC. IGF-1R, insulin like growth factor 1 receptor; miR, microRNA; 3′UTR, 3′-untranslated regions; WT, wild-type; MUT, mutant; NC, negative control.

miR-539 represses the AKT and ERK signaling pathways

Previous studies have demonstrated that IGF-1R serves important roles in biological processes associated with the downstream phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/ERK signaling pathways (29,30). The present study further determined whether miR-539 is involved in the AKT and EKR signaling pathways. Western blotting was performed to detect ERK, p-ERK, AKT and p-AKT expression in T24 cells following transfection with miR-539 mimics or miR-NC. As shown in Fig. 4, p-ERK and p-AKT were downregulated in miR-539 mimics-transfected T24 cells (P<0.05), however, miR-539 expression did not affect ERK and AKT expression. These results suggested that miR-539 may inhibit bladder cancer cell proliferation and invasion by activating the AKT and ERK signaling pathways.

Figure 4. miR-539 in the ERK and AKT signaling pathways. Western blotting was performed to detect ERK, p-ERK, AKT and p-AKT expression in T24 cells following transfection with miR-539 mimics or miR-NC. Upregulation of miR-539 suppressed p-ERK and p-AKT expression in T24 cells, however, ERK and AKT were not changed. The results were normalized to those of GAPDH. *P<0.05 vs. miR-NC. miR, microRNA; ERK, extracellular signal-regulated kinase; AKT, protein kinase B; p-, phosphorylated; NC, negative control.

Inverse correlation between miR-539 and IGF-1R in clinical bladder cancer tissues

To further elucidate the correlation between miR-539 and IGF-1R, the expression levels of IGF-1R mRNA and protein were detected in bladder cancer tissues and matched adjacent normal bladder tissues. The results revealed that IGF-1R mRNA and protein were significantly elevated in bladder cancer tissues in comparison to those observed in adjacent normal bladder tissues (P<0.05; Fig. 5A and B). In addition, IGF-1R mRNA expression was negatively correlated with the level of miR-539 expression in bladder cancer tissues (r=−0.5838, P<0.001; Fig. 5C).

Figure 5. miR-539 expression in bladder cancer tissues is negatively correlated with IGF-1R mRNA expression. IGF-1R (A) mRNA and (B) protein expression in bladder cancer tissues and matched adjacent normal bladder tissues were measured using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The western blotting results were normalized to those of GAPDH. The blotting images from 4 example cases are presented. *P<0.05 vs. matched adjacent normal bladder tissues. (C) Spearman's correlation analysis between miR-539 and IGF-1R mRNA expression levels in bladder cancer tissues. r=−0.5838; P<0.001. miR, microRNA; IGF-1R, insulin like growth factor 1 receptor.

miR-539 suppresses cell proliferation and invasion of bladder cancer by regulating IGF-1R

As IGF-1R is a direct target of miR-539, it was hypothesized that miR-539 may have tumor suppressive roles in bladder cancer via the regulation of its target, IGF-1R. To confirm this, si-IGF-1R was used to knockdown IGF-1R expression in T24 cells (P<0.05; Fig. 6A). The effects of IGF-1R knockdown on cell proliferation and invasion were evaluated using MTT and cell invasion assays, respectively. As shown in Fig. 6B and C, IGF-1R knockdown markedly suppressed the proliferation and invasion of T24 cells (P<0.05). These results indicated that the underexpression of IGF-1R induced by miR-539 may contribute, at least in part, to the suppression of bladder cancer cell proliferation and invasion.

Figure 6. IGF-1R knockdown inhibits the proliferation and invasion of T24 cells. (A) si-IGF-1R was used to knockdown IGF-1R expression in T24 cells, which was verified by western blotting. The results were normalized to those of GAPDH. (B) An MTT assay was performed to assess the proliferation of T24 cells transfected with si-IGF-1R or si-NC. Proliferation was significantly decreased in T24 cells transfected with si-IGF-1R following 96 h. (C) A cell invasion assay was performed to analyze the invasive capacity of T24 cells transfected with si-IGF-1R or si-NC (magnification, ×200). *P<0.05 vs. si-NC. IGF-1R, insulin like growth factor 1 receptor; si-, small interfering RNA; NC, negative control.

Discussion

Aberrant expression of miR-539 has been detected in a number of different types of human cancers. For example, in osteosarcoma, there is a low level of miR-539 expression in MG-63 cells when compared with osteoblast cells (31). Jin and Wang (32) demonstrated that miR-539 was downregulated in osteosarcoma tissues and cell lines. In addition, a previous study by Mirghasemi et al (19) revealed that low miR-539 expression was correlated with advanced Tumor, Node and Metastasis staging, and metastasis or recurrence in patients with osteosarcoma. Kaplan-Meier survival analysis and a log-rank test revealed that a low expression of miR-539 was significantly associated with a reduction in the overall survival rate of patients with osteosarcoma. The Multivariate Cox proportional hazards model demonstrated that decreased miR-539 expression was an independent prognostic marker of overall survival in patients with osteosarcoma (19). Gu and Sun (20) reported that miR-539 expression levels were reduced in thyroid cancer tissues and cell lines when compared with the respective control. In addition, in nasopharyngeal carcinoma, miR-539 levels decreased in tumor tissues when compared with normal tissues (21). These findings suggested that miR-539 may be an effective diagnostic and prognostic marker in these types of cancer.

Dysregulation of miR-539 is thought to contribute to the malignant phenotype of several types of tumor. In thyroid cancer, enforced expression of miR-539 suppressed cell migration and invasion by negatively regulating caspase recruitment domain-membrane-associated guanylate kinase protein 1 (20). In osteosarcoma, upregulation of miR-539 decreased cell growth and metastasis by directly targeting matrix metalloproteinase 8 (32). Lv et al (21) demonstrated that ectopic expression of miR-539 repressed cell growth in vitro and in vivo via a blockade of cyclin-dependent kinase 4. Zhang et al (33) reported that, in prostate cancer, miR-539 targeted sperm associated antigen 5 to inhibit cell proliferation, migration and invasion in vitro, and suppress tumor growth and metastasis in vivo. These findings indicated that miR-539 may act as a novel therapeutic target for the treatment of these types of cancer.

The present study revealed that miR-539 re-expression inhibited cell proliferation and invasion in bladder cancer. Subsequently, the potential molecular mechanism underlying the miR-539-induced inhibition of bladder cancer cell proliferation and invasion was determined. An important molecular association between miR-539 and IGF-1R was observed in bladder cancer. Initially, bioinformatics analysis predicted that IGF-1R contained a miR-539 seed match at the 3′UTR of IGF-1R. The luciferase reporter assay further demonstrated that miR-539 directly targeted the IGF-1R 3′UTR. RT-qPCR and western blot analysis was then performed and revealed that miR-539 negatively regulated IGF-1R expression in bladder cancer cells at the mRNA and protein level. IGF-1R mRNA was significantly upregulated in bladder cancer tissues and was negatively correlated with miR-539 level. In addition, IGF-1R knockdown suppressed cell proliferation and invasion, similar to the effect of miR-539 overexpression in bladder cancer cells. Identification of miR-539 targets is essential for understanding its role in bladder cancer formation and progression. In addition, it is important for developing novel therapeutic targets for the treatment of patients with bladder cancer.

IGF-1R, a transmembrane tyrosine kinase receptor of the insulin receptor family, contains two extracellular α subunits with a ligand-binding site and two transmembrane β subunits with intracellular tyrosine kinase activity (34). The IGF-1R itself has been frequently observed to be upregulated in various types of human cancers, including bladder cancer (24,35). In addition, IGF-1R expression in bladder cancer was significantly correlated with tumor grade, stage and recurrence (24). Furthermore, there is a growing body of evidence that supports the biological roles of IGF-1R in promoting tumorigenesis and progression of bladder cancer. Sun et al (25) reported that downregulation of IGF-1R suppressed the growth of T24 cells, induced apoptosis and improved cell chemosensitivity to mitomycin. Metalli et al (26) demonstrated that IGF-1R enhanced cell motility and invasion in bladder cancer. These results suggested that targeting IGF-1R could serve as a novel therapeutic method in bladder cancer.

To the best of our knowledge, this is the first study to provide experimental evidence that miR-539 was downregulated in bladder cancer, and its expression was associated with tumor stage and lymph node metastasis in patients with bladder cancer. Restoration of miR-539 expression inhibits bladder cancer cell proliferation and invasion. In addition, IGF-1R was identified as a direct target of miR-539 and miR-539 was observed to regulate the AKT and ERK signaling pathways. This newly identified miR-539/IGF-1R pathway may provide novel insights into the initiation and progression of bladder cancer, and may serve as a potential therapeutic target for the treatment of patients with bladder cancer.

References