RB tissues were obtained from 23 patients (age range, 15–34 years; average age, 23 years; 14 males and 9 females) who were diagnosed with RB and treated via surgical resection at the China-Japan Union Hospital of Jilin University (Changchun, China). Normal retinal tissues were collected from seven patients (five males and two females) suffering from globe rupture. Their ages ranged between 28 and 63 years, and the average age was 41 years. All tissues were obtained between March 2015 and January 2017. Patients that had been treated with laser therapy, cryotherapy, thermotherapy, chemotherapy or radiotherapy were excluded from the present study. All tissues were immediately frozen in liquid nitrogen and subsequently stored at −80°C until further extraction of protein or RNA. The present study was approved by the Ethics Committee of China-Japan Union Hospital of Jilin University. Written informed consent was provided by all enrolled patients.

A total of three RB cell lines (Y79, SO-RB50 and Weri-RB1) and the normal retinal pigmented epithelial cell line ARPE-19, used as the control in the present study, were purchased from The American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and grown at 37°C in a humidified incubator containing 5% CO₂.

The miR-504 mimics and negative control miRNA (miR-NC) were chemically synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The miR-504 mimics sequence was 5′-AGACCCUGGUCUGCACUCUAUC-3′ and the miR-negative control (NC) sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. An astrocyte elevated gene-1 (AEG-1)-overexpressing plasmid was constructed using a pcDNA3.1 (+) basic plasmid supplied by Shanghai GenePharma Co., Ltd. (Shanghai, China), and was defined as pcDNA3.1-AEG-1. The restriction sites were NheI and HindIII. An empty pcDNA3.1 vector was used as the control for pcDNA3.1-AEG-1 transfection. Y79 and Weri-RB1 cells were transfected with miRNA mimics (100 pmol) or plasmid (4 µg) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Co-transfection of miR-504 mimics and pcDNA3.1-AEG-1 was additionally performed using Lipofectamine^® 2000. After 8 h, the culture medium was replaced with fresh DMEM containing 10% FBS. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were conducted after 48 and 72 h incubations, respectively. Cell Counting Kit-8 (CCK-8) and cell invasion assays were conducted at 24 and 48 h post-transfection, respectively.

Total RNA was extracted from tissues or cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. For the detection of miR-504 expression, cDNA was produced using the TaqMan^® MicroRNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The RT temperature protocol was as follows: 16°C for 30 min, 42°C for 30 min and 85°C for 5 min. qPCR was subsequently performed using the TaqMan MicroRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The thermocycling conditions were as follows: 50°C for 2 min, 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 sec; and annealing/extension at 60°C for 60 sec. For the quantification of AEG-1 mRNA expression, total RNA was reverse transcribed with the RevertAid™ First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). The RT temperature protocol was as follows: 37°C for 5 min, 42°C for 60 min, 70°C for 10 min and left on ice. The synthesized cDNA was subjected to qPCR using the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc.). The thermocycling conditions for qPCR 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 small nuclear RNA and GAPDH were utilized as endogenous controls for miR-504 and AEG-1 mRNA, respectively. All reactions were performed on the Applied Biosystems 7500 real-time PCR system (Thermo Fisher Scientific, Inc.). Each assay was performed in triplicate and repeated three times. The 2^−ΔΔCq method was used to analyze gene expression (23). The primers were designed as follows: miR-504, 5′-AGACCCTGGTCTGCACTCTAT-3′ (forward) and 5′-GCGAGCACAGAATTAATACGAC-3′ (reverse); U6, 5′-CAAGGATGACACGCAAAT-3′ (forward) and 5′-GCGAGCACAGAATTAATACGAC-3′ (reverse); AEG-1, 5′-TGTTGAAGTGGCTGAGGG-3′ (forward) and 5′-CAGGAAATGATGCGGTTG-3′ (reverse); and GAPDH, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ (forward) and 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′ (reverse).

Transfected Y79 and Weri-RB1 cells were collected following 24 h incubation, and were inoculated into 96-well plates at a density of 3,000 cells/well. A CCK-8 assay was conducted at four time points: 0, 24, 48 and 72 h following inoculation. At each time point, 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added into each well, and the cells were incubated for an additional 2 h at 37°C. The absorbance value of each well was detected at a wavelength of 450 nm by a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

In vitro cell invasion assays were performed in order to measure the invasive ability of RB cells using Transwell chambers (8-µm pore size) precoated with Matrigel (both from BD Biosciences, San Jose, CA, USA). Transfected Y79 and Weri-RB1 cells were collected following 48 h of incubation at 37°C, resuspended in FBS-free DMEM and plated into the upper chambers with an initial density of 5×10⁴ cells/chamber. The lower chambers were coated with 500 µl DMEM containing 20% FBS. After 24 h incubation at 37°C, cells that had not invaded via the pores were gently removed using a cotton swab, whereas the invasive cells were fixed with 4% paraformaldehyde at 37°C for 30 min and stained with 0.1% crystal violet at 37°C for 30 min. Images of the invasive cells were captured and the number of invasive cells was counted in at least five randomly selected visual fields under a light microscope (magnification, ×200; Olympus IX83; Olympus Corporation, Tokyo, Japan).

TargetScan (Release 7.2; http://www.targetscan.org/vert_71/) and miRanda (Release Last Update: 2010-11-01; http://34.236.212.39/microrna/home.do) were used to predict the putative targets of miR-504.

For the reporter assay, the plasmids pmirGLO-AEG-1-3′-UTR wild-type (wt) and pmirGLO-AEG-1-3′-UTR mutant (mut), respectively containing the wt and mut miR-504 binding site in the 3′-UTR of AEG-1, were chemically synthesized by Shanghai GenePharma Co., Ltd. Y79 and Weri-RB1 cells were seeded into 24-well plates at a density of 1.0×10⁵ cells/well one night prior to transfection. pmirGLO-AEG-1-3′-UTR wt or pmirGLO-AEG-1-3′-UTR mut was transfected into cells using Lipofectamine^® 2000 in the presence of miR-504 mimics or miR-NC. Transfected cells were harvested at 48 h following incubation, and the luciferase activity was measured using the Dual-Luciferase Reporter System (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. The firefly luciferase activity of each sample was normalized to that of the Renilla luciferase activity.

The expression of AEG-1 protein was detected by western blot analysis. Radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) was applied to isolate total protein from tissues or cells. Protein expression was quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Equal amounts of total protein (30 µg) were loaded, separated by 10% SDS-PAGE and subsequently transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% skimmed milk diluted in Tris-buffered saline containing 0.1% Tween 20 buffer (TBST) at room temperature for 1 h, and further incubated overnight at 4°C with the following antibodies: Mouse anti-human AEG-1 monoclonal antibody (1:1,000 dilution; cat. no. sc-517220; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse anti-human GAPDH monoclonal antibody (1:1,000 dilution; cat. no. sc-166574; Santa Cruz Biotechnology, Inc.). Subsequently, the membranes were washed three times with TBST and probed with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution; cat. no. sc-516102; Santa Cruz Biotechnology, Inc.) at 37°C for 1 h. The protein signals were observed using an enhanced chemiluminescence kit (EMD Millipore) according to the manufacturer's protocol. Quantity One software (version 4.62; Bio-Rad Laboratories, Inc., Hercules, CA, USA) was utilized for quantification of densitometry.

All statistical analysis was performed using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). All data are presented as the mean ± standard deviation from at least three independent experiments. A Student's two-tailed t-test and one-way analysis of variance (ANOVA) was employed to compare the differences between groups. A Dunnett's test was used as a post hoc test following ANOVA. Spearman's correlation analysis was utilized to analyze the association between miR-504 and AEG-1 in RB tissues. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the expression status of miR-504 in RB, the expression levels of miR-504 in 23 RB and seven normal retinal tissues were analyzed. The results of RT-qPCR demonstrated that miR-504 expression levels were significantly lower in RB tissues compared with the normal retinal tissues (P<0.05; Fig. 1A). Subsequently, miR-504 expression in three RB cell lines was measured using RT-qPCR. miR-504 expression was significantly downregulated in all three RB cell lines (Y79, SO-RB50 and Weri-RB1) compared with in the normal retinal pigmented epithelial cell line ARPE-19 (P<0.05; Fig. 1B). The results indicated that the dysregulation of miR-504 may be involved in the development and progression of RB.

To investigate the specific roles of miR-504 in RB, miR-504 mimics or miR-NC were transfected into Y79 and Weri-RB1 cells, which exhibited notably lower miR-504 expression levels compared with SO-RB50 cells. Following transfection, RT-qPCR was utilized to assess the transfection efficiency, which revealed that transfection of miR-504 mimics significantly increased the levels of miR-504 in Y79 and Weri-RB1 cells (P<0.05; Fig. 2A). The results of the CCK-8 assay demonstrated that exogenous miR-504 expression significantly decreased the proliferation of Y79 and Weri-RB1 cells compared with the control (P<0.05; Fig. 2B). In vitro cell invasion assays were subsequently employed to determine the effect of miR-504 upregulation on the invasive ability of RB cells. It was demonstrated that miR-504 restoration was able to significantly suppress the invasion of Y79 and Weri-RB1 cells compared with the control (P<0.05; Fig. 2C). From the CCK-8 assay, it was observed that the inhibitory effects of miR-504 on RB cell proliferation had no statistical significance at 24 h following incubation. In vitro cell invasion assays were performed in Y79 and Weri-RB1 cells following 24 h incubation. Thus, it was proposed that the reduced invasive potential may be due to anti-invasive mechanisms rather than reduced cell number. The results demonstrated that miR-504 may exert tumor-suppressor activity in RB.

To understand the mechanisms underlying the proliferation and invasion suppression by miR-504 in RB cells, bioinformatics analysis was applied to determine potential miR-504 targets. As presented in Fig. 3A, it was identified that the 3′-UTR of AEG-1 contains a binding site for miR-504. AEG-1 was selected for further experimental verification as the gene has been reported to be closely associated with the genesis and development of RB (24). A luciferase reporter assay was adopted to confirm whether miR-504 could directly target the 3′-UTR of AEG-1. It was observed that miR-504 upregulation significantly attenuated the luciferase activity of the plasmid containing wt AEG-1 3′-UTR in Y79 and Weri-RB1 cells compared with the control (P<0.05); however, the luciferase activity of the plasmid harboring the mutated binding site was markedly unaffected (Fig. 3B). Via RT-qPCR and western blot analysis, AEG-1 mRNA and protein levels were examined in Y79 and Weri-RB1 cells transfected with miR-504 mimics or miR-NC; whether miR-504 affects endogenous AEG-1 expression was also investigated. Overexpression of miR-504 significantly suppressed AEG-1 mRNA (P<0.05; Fig. 3C) and protein (P<0.05; Fig. 3D) expression levels in Y79 and Weri-RB1 cells compared with the control. The reported results indicated that miR-504 directly targeted the 3′-UTR of AEG-1 and inhibited its expression in RB cells.

AEG-1 was predicted as a direct target gene of miR-504 in RB. Thus, AEG-1 expression was detected in RB tissues, and a potential association between miR-504 and AEG-1 expression was evaluated. The data from RT-qPCR analysis revealed that the mRNA expression levels of AEG-1 were significantly higher in RB tissues compared with normal retinal tissues (P<0.05; Fig. 4A). Spearman's correlation analysis was performed to further demonstrate the association between miR-504 and AEG-1 mRNA expression in RB tissues. The analysis revealed a negative correlation between miR-504 and AEG-1 mRNA levels in RB tissues (r=−0.5463; P=0.0070; Fig. 4B), suggesting that the upregulation of AEG-1 in RB tissues may be partly attributed to miR-504 downregulation.

In order to demonstrate that miR-504 inhibits RB cell proliferation and invasion by suppressing AEG-1 expression, whether the reported effects could be rescued by upregulating AEG-1 was investigated. Y79 and Weri-RB1 cells were co-transfected with miR-504 mimics and AEG-1 overexpression plasmid (pcDNA3.1-AEG-1) or empty pcDNA3.1, and the transfected cells were subsequently subjected to a series of functional assays. Western blot analysis confirmed that co-transfection with pcDNA3.1-AEG-1 successfully eliminated the miR-504 overexpression-induced inhibition of AEG-1 protein expression in Y79 and Weri-RB1 cells (P<0.05; Fig. 5A). Subsequently, CCK-8 and in vitro cell invasion assays demonstrated that the tumor-suppressing roles of miR-504 in Y79 and Weri-RB1 cell proliferation (P<0.05; Fig. 5B) and invasion (P<0.05; Fig. 5C) were mitigated by AEG-1 overexpression. Collectively, the results of the present study suggest that miR-504 exerts its antitumor effects in RB, at least partly, via the negative regulation of AEG-1.