The etiology and pathogenesis of bladder cancer (BCa) is complex. MicroRNA (miRNA) has been implicated in BCa. Targeting of signal transducer and activator of transcription 3 (STAT3) by miR-124 to regulate tumorigenesis has been demonstrated in other types of cancer. In the present study, miR-124 levels were downregulated in the BCa T24 cell line and STAT3 was increased in BCa cell lines. Transfection of miR-124 mimics into T24 cells significantly inhibited STAT3 expression. A luciferase assay confirmed that miR-124 directly targeted the STAT3 3′untranslated region to inhibit STAT expression. Knockdown of STAT3 expression led to increased apoptosis of T24 cells and reduced tumor growth
Bladder cancer (BCa) is one of the most common genitourinary malignancies worldwide that causes an estimated 150,000 mortalities annually globally (
MicroRNAs (miRNAs) are small (22 nucleotide long) noncoding RNA molecules that are highly conserved in eukaryotes (
The present study demonstrated a downregulation of miR-124 in T24 BCa cells that was inversely associated with STAT3 expression. Furthermore, miR-124 inhibited STAT3 expression by directly targeting its 3′UTR. Knockdown of STAT3 significantly promoted apoptosis and suppressed cell cycle progression, migration, proliferation, and colony formation in T24 cells. The collective results suggest that miR-124 may directly inhibit the expression of STAT3 in BCa, and that miR-124 may suppress BCa tumorigenesis by targeting STAT3.
Human BCa T24, UM-UC-3, SW780, HT1376, RT4 and J82 cell lines, immortalized human bladder epithelium SV-HUC-1 cells and the 293 cell line were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured according to the manufacturer's instructions at 37°C in an atmosphere of 5% CO2. Total mRNA and miRNA were independently extracted from the cells using RNAzol RT RNA Isolation Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and stored at −20°C for subsequent analysis.
The expression of miR-124 was quantified using an All-in-one miRNA qPCR kit (GeneCopoeia, Inc., Rockville, USA) and U6 was used as the control. The primers used for the RT-PCR were: miR-124 (forward 5′-GCGGCCGTGTTCACAGCGGACC-3′ and reverse 5′-GTGCAGGGTCCGAGGT-3′) and U6 (forward 5′-CGCTTCGGCAGCACATATACTA-3′ and reverse 5′-CGCTTCACGAATTTGCGTGTCA-3′). To determine the mRNA expression of STAT3, the total RNA in T24 cells was reverse transcribed into cDNA at 37°C using Eastep RT Master Mix kit (Promega Corporation, Madison, WI, USA), then STAT3 mRNA was quantified using SYBR Premix Ex Taq (Takara Bio, Inc., Otsu, Japan) and GAPDH was used as the control. The thermocycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 60°C for 34 sec, then 72°C for 45 sec. The primers used for qPCR were: STAT3 (forward 5′-ATCACGCCTTCTACAGACTGC-3′ and reverse 5′-CATCCTGGAGATTCTCTACCACT-3′) and GAPDH (forward 5′-CCACTCCTCCACCTTTGAC-3′ and reverse 5′-ACCCTGTTGCTGTAGCCA-3′). Relative expression was calculated using the 2−ΔΔCq method (
T24 cells were centrifuged in 1,000 × g for 5 min at 37°C subsequent to the addition of 1 ml ice-cold PBS and washed three times with ice-cold PBS and lysed using a Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology, Haimen, China) for 30 min at 4°C. The cell lysate protein was centrifuged at 15,000 × g at 4°C for 10 min and the protein was quantified using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). A total of 20 µg protein/lane was separated by 10% SDS-PAGE, and the resolved proteins were transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked using 5% skim milk powder at 4°C overnight and then hybridized with the primary antibodies targeting the proteins including GADPH (1:2,500; cat no. ab9485), STAT3 (1:1,000; cat no. ab68153), vascular endothelial growth factor receptor (VEGFR; 1:1,000; cat no. ab11939), B-cell lymphoma 2 (Bcl-2; 1:1,000; cat no. ab32124), B-cell lymphoma-extra large (Bcl-xl; 1:1,000; cat no. ab32370), MCL1, Bcl2 family apoptosis regulator (Mcl-1; 1:2,000; cat no. ab32087), Cyclin D1 (1:10,000; cat no. ab134175) and MYC proto-oncogene, BHLH transcription factor (cMyc; 1:1,000; cat no. ab32072) (all from Abcam, Cambridge, UK) at 4°C overnight. Subsequent to washing using PBS with 0.05% Tween-20 three times at 5 min each time, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibody Goat Anti-Rabbit immunoglobulin G H&L (1:2,000; cat no. ab6721; Abcam) conjugated to horseradish peroxidase at 4°C overnight. Results were quantified by the Molecular Imager ChemiDoc XRS+ System 2.0 (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH was used as the control.
The whole 3′UTR of the STAT3 gene was cloned and amplified in 293 cells. TargetScan 6.2 bioinformatics prediction software (
T24 (1×105 cells/plate) were plated in 6-well plates at 37°C overnight and transfected with miR-124 mimics or NC mimics (20 nM; GeneCopoeia, Inc.) using Lipofectamine® 2000. The miR-124 mimic sequence used was: Forward, 5′-GCTCTAGAGGCCTCTCTCTCCGTGTTCCACAGCGGACCTTGATTTAAATGTCCATACAATTAAGGCACGCGGTTGAATGCCAAGAATGGGGCTG-3′ and reverse, 5′-CGGGATCCCAGCCCCATTCTTGGCATTCACCGCGTGCCTTAATTGTATGGACATTTAAATCAAGGTCCGCTGTGAACACGGAGAGAGAGGCCT-3′. Following transfection for 6 h at 25°C, the Opti-MEM medium (Gibco; Thermo Fisher Scientific, Inc.) without serum was changed with the fresh medium. Then cells were assayed by RT-qPCR and western blot analysis for each group according to the aforementioned protocols following culture for 48 h.
Small interfering RNA Target Finder online design software (Ambion; Thermo Fisher Scientific, Inc.) was used to select a segment (5′-AAGAGTCAAGGAGACATGCAA-3′, 670–690 bp) in the coding region of STAT3 (GenBank serial no. NM139276) as a target sequence. Then, the corresponding DNA template strands containing restriction sites for
The adherent cells were digested at 37°C with 0.25% trypsin (Bioswamp; Wuhan Myhalic Biotechnological Co., Ltd., Wuhan, China) and centrifuged at 1,000 × g at 25°C for 5 min. The supernatant was discarded and the cells were washed three times with PBS. The cells (1–5×105) were collected and resuspended in 200 µl binding buffer (cat no. C1052-1; Beyotime Institute of Biotechnology) for analysis using the Cell Cycle and Apoptosis Analysis kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Analyses were performed using a LSRII Flow Cytometry System equipped with FACSDiva software 4.1 (BD Biosciences, Franklin Lakes, NJ, USA). Data was analyzed with the ModFit LT software package version 4.0 (Verity Software House, Inc., Topsham, ME, USA).
The WST-1 Cell Proliferation and Cytotoxicity Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol was used to analyze cell viability and cell proliferation at 0, 24, 48, 72 and 96 h. All experiments were performed in triplicate. For the colony formation assay, cells were plated at a density of 200 cells/well in standard culture conditions (5% CO2 and 37°C) for 14 days. The colonies were fixed with 5 ml 4% absolute methanol for 15 min at 25°C, stained at 37°C with 0.1% crystal violet for 20 min and washed three times. Then a light microscope (TS-100F; Nikon, Tokyo, Japan) was used to visualize the results at a magnification of ×40.
T24 cells were seeded (1×106 cells/plate) in a 6-well plate. Wounding of the confluent cell growth was performed by scratching with a 10 µl pipette tip 24 h after cell plating. The cells were then cultured for 24 h and wound closure was detected by inverted microscopy (magnification, ×200; Olympus, Tokyo, Japan). The experiment was repeated three times.
Statistical analysis was conducted using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Unpaired Student's t-tests were used to analyze the results. Pearson's linear correlation analysis was used to analyze the association between the expression of STAT3 and miR-124. All experiments were performed in triplicate and the data are expressed as mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.
To evaluate the function of STAT3 in human BCa, the expression of STAT3 was detected in 6 human BCa cell lines (T24, UM-UC-3, SW780, HT1376, RT4 and J82) and immortalized human bladder epithelium SV-HUC-1 cells. STAT3 was overexpressed as an oncogene in the BCa cell lines compared with SV-HUC-1 cells. The expression of STAT3 was highest in T24 cells (
The effects of the transfection of miR-124 mimics on STAT3 mRNA and protein expression levels were determined by RT-qPCR and western blot analysis. There was an ~80% decrease in STAT3 mRNA expression and a 60% decrease in STAT3 protein expression in T24 cells transfected with miR-124 mimics (
To additionally investigate the role of miR-124/STAT3 signaling pathway in BCa, a stable cell line with low STAT3 expression (STAT3-shRNA-T24) and a control cell line (shRNA-control-T24) were constructed (
To investigate the effect of STAT3 on the migration of T24 BCa cells, the capacity of STAT3-shRNA-T24 cells to migrate was measured using a wound healing assay. STAT3-shRNA-T24 cells exhibited slower rates of wound closure and decreased numbers of stained cells in the wound healing assay compared with the shRNA-control-T24 cells (
miRNA has been studied intensively since it was suggest to be associated with cancer in 2002 (
STAT3 is a recognized oncogene that belongs to the STAT family. In tumors, it is generally always stimulated by a variety of extracellular signals that include growth factors and cytokines (
In conclusion, the data of the present study indicate a novel role of the miR-124/STAT3 signaling pathway in BCa and demonstrate the potential to use miR-124 or STAT3 as a diagnostic marker or therapeutic tool for human BCa. However, there were several limitations in the present study. The study was validated in only one bladder cancer cell line¸ T24, therefore the results of this study suggest that this may be cell-type specific. Therefore, the association between miR-124 and STAT3
Not applicable.
The present study was supported by grants from Project of Hainan Natural Science Foundation of China (grant no. 20168304) and Project capital of Hainan Provincial Department of health (grant no. 2013 self raising-10).
All datasets used during the current study are available from the corresponding author on reasonable request.
SW was responsible for the project and performed the experiments. PL lead experimental work and conducted immunohistochemistry work; GW actualized the fluorescence detection. YH conducted the fluorescence detection and specimen treatment. The immunohistochemistry experiments were performed by PS, JC and YW. JY implemented autofluorescence acquisition and software system processing.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Relevance of miR-124 and STAT3 in bladder cancer. (A) The expression of STAT3 protein in bladder cancer cell lines. (B) Relative expression levels of miR-124 and STAT3. (C) Reverse transcription quantitative polymerase chain reaction was used to detect the level of STAT3 mRNA in T24 cell lines following transfection of NC mimics and miR-124 mimics for 48 h. (D) Western blot analysis was used to detect the expression of STAT3 protein in T24 cell lines following transfection of NC mimics and miR-124 mimics for 48 h. (E) Schematic construction of wild-type or mutant miR-124 binding sequences in the STAT3 3′-UTR vector. The mutant binding sequences are in red. (F) Suppressed luciferase activity of wild-type 3′-UTR of STAT3 by pre-miR-124. All data are presented as the mean ± standard deviation of the mean. **P<0.01 and ***P<0.001. miR, microRNA; STAT3, signal transducer and activator of transcription 3; NC, negative control; UTR, untranslated region; CMV promotor, cytomegalovirus promotor; WT, wild type; MUT, mutant.
Construction and identification of STAT3-shRNA stably-transfected cells. (A) Fluorescence images of shRNA-transfected T24 cells (magnification, ×200). (B) Reverse transcription quantitative polymerase chain reaction was used to detect the level of STAT3 mRNA in STAT3-shRNA-T24 cell lines. (C) Western blot analysis was used to detect the expression of STAT3 protein in STAT3-shRNA-T24 cell lines. All data are presented as the mean ± standard deviation of the mean. **P<0.01. shRNA, short hairpin RNA; STAT3, signal transducer and activator of transcription.
Knockdown of STAT3 significantly suppresses cell cycle progression, proliferation and colony formation in T24 cells. (A) The effects of knockdown of STAT3 on cell cycle in T24 cells were analyzed by flow cytometry. A total of three independent experiments were performed in each group. (B) Cell viability was examined using an WST-1 assay. (C) Effect of knockdown of STAT3 on the proliferation rate in T24 cells was measured by a colony formation assay. All data are presented as the mean ± standard deviation of the mean. *P<0.05, **P<0.01 and ***P<0.001. shRNA, short hairpin RNA; STAT3, signal transducer and activator of transcription.
Knockdown of STAT3 significantly promotes apoptosis and suppresses cell migration and STAT3 downstream target genes in T24 cells. (A) The effects of knockdown of STAT3 on migration in T24 were analyzed using wound healing assays with a 24 h recovery period. (B) Apoptosis was determined by fluorescence-activated cell sorting. Cells in early apoptosis are in the bottom right quadrant, while cells in late apoptosis are in the top right quadrant. (C) Western blot analysis was used to detect the expression of STAT3 downstream target proteins. All data are presented as the mean ± standard deviation of the mean. *P<0.05 and **P<0.01. shRNA, short hairpin RNA; STAT3, signal transducer and activator of transcription; PI, propidium iodide; FITC, fluorescein isothiocyanate; VEGFR, vascular endothelial growth factor receptor; Bcl-2, B-cell lymphoma 2; Bcl-xl, B-cell lymphoma-extra large; Mcl-1, MCL1, Bcl2 family apoptosis regulator; cMyc, MYC proto-oncogene, BHLH transcription factor.