A total of 30 RB and adjacent normal tissues were collected from 30 patients with RB (age range 0–7 years; 12 females and 18 males) at the First People's Hospital of Kunshan Affiliated with Jiangsu University (Suzhou, China) from May 2015 to May 2017. All of the 30 RB patients received enucleation or enucleation with chemotherapy and with or without radiotherapy. The present study was approved by the Ethical Committee of the First People's Hospital of Kunshan Affiliated with Jiangsu University, and informed consent was obtained from all patients.

The RB cell lines, Y79, WERI-RB1, SO-RB50 and SO-RB70, and retinal pigment epithelial cell line, ARPE-19, were purchased from the Shanghai Institute of Life Sciences at the Chinese Academy of Sciences (Shanghai, China). Y79, WERI-RB1, SO-RB50 and SO-RB70 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). The ARPE-19 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. All cells were incubated in a humidified incubator at 37°C with 5% CO₂.

Y79 cells were treated with (50, 100, 500 or 1,000 µM) lidocaine (MedChem Express, MCE, USA) at 37°C for 12, 24, or 48 h respectively. Cells without any treatment were used as the control group.

Y79 cells were seeded into 6-well plates 1 day before transfection and incubated at 37°C with 5% CO₂. When the cell confluence reached 60–70%, the Y79 cells were transfected with 100 nM miR-520a-3p inhibitor (inhibitor) (cat. no. miR20002834-1-5; Guangzhou RiboBio Co., Ltd., Guangzhou, China), 100 nM miR-520a-3p mimic (mimic) (cat. no. miR10002834-1-5; Guangzhou RiboBio Co., Ltd.) or 100 nM miR-520a-3p scramble [negative control (NC; cat. no. miRB160401025525-2-1; Guangzhou RiboBio Co., Ltd.) using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.)] for 24 h according to the manufacturer's protocols. Then, 24 h after cell transfection, subsequent experiments were performed.

A CCK-8 assay was performed to determine the viability of Y79 cells. Logarithmic phase Y79 cells were plated in 96-well plates (1×10⁴ cells/well) and treated with (50, 100, 500 or 1,000 µM) lidocaine, then the cells were incubated in an 37°C, 5% CO₂ incubator for 12, 24, or 48 h respectively. Subsequently, 10 µl CCK-8 reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to every well and cells were incubated at 37°C with 5% CO₂ for a further 2 h. To determine cell proliferation, a microplate reader was used to detect the absorbance at 450 nm.

Y79 cells were collected in the logarithmic growth phase and inoculated into 6-well plates at 1×10⁵ cells/well. Y79 cells were then treated with 500 or 1,000 µM lidocaine at 37°C for 24 or 48 h (or Y79 cells were transfected with/without miR-520a-3p inhibitor or NC at 37°C for 24 h, and the cells were then treated with/without 500 µM lidocaine at 37°C for 24 h). Subsequently, an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (cat. no. 70-AP101-100; MultiSciences Biotech, Co., Ltd., Hangzhou, China) was used to determine the number of apoptotic cells, according to the manufacturer's protocols. Collected cells were stained with 0.5 ml of binding buffer plus Annexin V-FITC and PI at room temperature for 15 min in the dark. A flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was then used to analyze the apoptosis of cells. The number of cells undergoing early and late apoptosis were determined using WinMDI soft-ware (version 2.5; Purdue University Cytometry Laboratories; www.cyto.purdue.edu/flowcyt/software/Catalog.htm) and the apoptosis rate was presented.

TargetScan 7.2 (http://www.targetscan.org/vert_72/) was used to predict the potential targets of miR-520a-3p, and the binding sites between miR-520a-3p and EGFR were observed. To investigate the association between miR-520a-3p and EGFR, luciferase reporter plasmids was generated containing the 3′-UTR sequence of EGFR. Wild-type (WT) and mutant (MUT) 3′-untranslated regions (UTRs) of EGFR were amplified by PCR using DreamTaq DNA Polymerase (Thermo Fisher Scientific, Inc.) and then cloned into the psiCHECK-2 reporter (cat no. C8011; Promega Corporation, Madison, WI, USA). Y79 cells were co-transfected with mimic or NC and the mutant (MUT-EGFR) or wild-type (WT-EGFR) 3′-UTR of EGFR and 25 ng pRL-TK (expressing Renilla luciferase as the internal control; Promega Corporation) using Lipofectamine^® 2000 reagent for 48 h. Luciferase activity was determined 48 h after cell transfection using the Dual-Luciferase Assay system (Promega Corporation,) on a luminometer (Mithras LB940; Berthold Technologies USA, LLC, Oak Ridge, TN, USA) according to the manufacturer's protocols and normalized to Renilla luciferase activity. Experiments were repeated in triplicate.

Total RNA from tissues and cells was collected using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using PrimeScript RT reagent kit (Takara Bio, Inc.) according to the manufacturer's protocols. qPCR was performed using the cDNA the SYBR RT-PCR kit (Takara Bio, Inc.). The thermocycling conditions were as follows: 95°C for 5 min, followed by 38 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 30 sec. Primer sequences were: U6, forward 5′-GCTTCGGCAGCACATATACTAAAAT-3′; reverse 5′-CGCTTCACGAATTTGCGTGTCAT-3′; GAPDH, forward 5′-CTTTGGTATCGTGGAAGGACTC-3′; reverse 5′-GTAGAGGCAGGGATGATGTTCT-3′; miR-520a-3p, forward 5′-ACACTCCAGCTGGGAAAGTGCTTCCC-3′; reverse 5′-CTCAACTGGTGTCGTGGA-3′; EGFR, forward 5′-CGGGACATAGTCAGCAGTG-3′; reverse 5′-GCTGGGCACAGATGATTTTG-3′.

The 2^−ΔΔCq method (28) was used to determine the relative gene expression normalized to GAPDH (for mRNA) or U6 (for miR-520a-3p). Experiments were repeated three times.

Protein expression was determined via western blotting. Proteins from cells or tissues were obtained using a modified radioimmunoprecipitation assay buffer (Auragene Bioscience, Changsha, China) with 1 mM phenylmethane sulfonyl fluoride for 20 min. A BCA protein quantitative kit (Thermo Fisher Scientific, Inc.) was used to analyze protein concentration. Equal quantities of lysate samples (25 µg/lane) were separated via 10% SDS-PAGE and transferred to 0.45 mm polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk at room temperature for 1.5 h and then incubated with primary antibodies: EGFR (cat. no. 4267; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) and β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. Following three washes with PBS-Tween-20, the membranes were subsequently incubated with the anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibody (cat no. 7074; anti-rabbit IgG, HRP-linked antibody; 1:5,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, SignalFire™ enhanced chemiluminescence reagent (cat. no. 6883; Cell Signaling Technology, Inc.) was used to visualize the protein bands. β-actin was used as the internal control.

All animals experiments were performed following the Recommended Guidelines for the Care and Use of Laboratory Animals issued by Chinese Council on Animal Research (29). The present study was approved by Animal Ethics Committee of the First People's Hospital of Kunshan Affiliated with Jiangsu University. A total of 50 specific pathogen-free male mice (5–6 weeks of age, 20–25 g) were purchased from the Model Animal Research Centre (Nanjing, China). The environment was maintained at a constant temperature (22–25°C) with 40–50% humidity and 12 h dark/light cycle conditions. All mice had access to food and water ad libitum. Single cell suspensions of Y79 cells were prepared in sterile PBS. Non-transfected tumor cells (2×10⁶), or cells transfected with 100 nM inhibitor or 100 nM NC for 24 h were injected subcutaneously into the flanks of nude mice. Mice were then administrated with lidocaine (1.5 mg/kg) (30) via tail vein injection, or an equal volume of sterile PBS as control treatment. Tumor growth was monitored twice per week and the volume of tumors were calculated using the following formula: Volume=(length × width²)/2. Additionally, analytical balances were used to determine the weight of tumors. Then, 18 days after tumor inoculation, RB and adjacent tissues from mice were collected for RT-qPCR/western blot analyses. The tumor experiments were ended when tumor diameter <20 mm.

All experiments were repeated three times. Data are presented as the mean ± standard deviation. Differences between groups were analyzed using unpaired or paired Student's t-tests or one-way analyses of variance followed by a Tukey's test. Data were analyzed using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the anti-tumor properties of lidocaine, a CCK-8 assay was performed using the human RB cell line, Y79. Y79 cells were treated with various concentrations of lidocaine (50, 100, 500 and 1,000 µM) (26,27) for 12, 24 and 48 h, and the viability of cells was determined. It was revealed that lidocaine (500 and 1,000 µM) significantly reduced the proliferation of Y79 cells at 24 and 48 h (Fig. 1). To further investigate whether lidocaine inhibited the proliferation of Y79 cells via the induction of apoptosis, flow cytometry was conducted. It was demonstrated that lidocaine (500 and 1,000 µM) increased the rate of the apoptosis of Y79 cells at 24 and 48 h (Fig. 2).

To determine the role of miR-520a-3p in RB, RT-qPCR analysis was performed to determine the relative expression of miR-520a-3p in 30 RB and adjacent normal tissues, plus Y79, WERI-RB1, SO-RB50 and SO-RB70 RB cells, and ARPE-19 retinal pigment epithelial cells. It was demonstrated that miR-520a-3p was significantly downregulated in RB tissues and cell lines compared with normal tissues and cells; expression was most notably decreased in Y79 cells (Fig. 3A and B). As lidocaine significantly induced the apoptosis of Y79 cells, Y79 cells were selected for further analysis. Y79 cells were treated with 500 µM lidocaine for 24 h, and RT-qPCR analysis was performed to determine the expression levels of miR-520a-3p. It was observed that lidocaine significantly upregulated the expression of miR-520a-3p in Y79 cells compared with the control (Fig. 3C).

TargetScan was used to identify functional targets of miR-520a-3p. Targets can results revealed that the 3′-UTR of EGFR contained a putative site that was partially complementary to miR-520a-3p (Fig. 4A). Luciferase reporter plasmids containing the WT-EGFR or MUT-EGFR were generated; a luciferase reporter assay demonstrated that mimic significantly reduced the luciferase activity of cells transfected with the WT-EGFR plasmid only, compared with the control (Fig. 4B). Y79 cells were transfected with NC or mimic, and the transfection efficiency was determined after 48 h. RT-qPCR and western blot analysis revealed that transfection with mimic significantly increased the expression of miR-520a-3p (Fig. 4C) and downregulated the expression of EGFR at the mRNA and protein levels compared with the control (Fig. 4D and E).

RT-qPCR and western blot analyses were conducted to investigate the expression of EGFR in RB tissues and cell lines. It was demonstrated that EGFR expression was significantly upregulated in RB tissues and cells compared with normal controls (Fig. 5A-D). Y79 cells were treated with 500 µM lidocaine for 24 h; it was revealed that lidocaine significantly suppressed the expression of EGFR in Y79 cells compared with the control (Fig. 5E and F).

Y79 cells were transfected with NC or inhibitor for 24 h to investigate the association between lidocaine and miR-520-3p. The cells were then treated with lidocaine or control for 24 h. It was revealed that inhibitor significantly suppressed the expression of miR-520a-3p and increased the levels of EGFR compared with the control (Fig. 6). Furthermore, compared with lidocaine treatment alone, the expression levels of miR-520a-3p were significantly decreased following treatment with lidocaine plus transfection with inhibitor, whereas those of EGFR were upregulated.

CCK-8 and flow cytometry assays were conducted to investigate the viability and apoptosis of cells following transfection with inhibitor and/or treatment with 500 µM lidocaine. Compared with the control group, inhibitor significantly promoted the proliferation and inhibited the apoptosis of Y79 cells. Additionally, compared with lidocaine treatment alone, combining transfection with inhibitor and lidocaine treatment significantly increased Y79 cell proliferation and reduced cell apoptosis (Fig. 7). The results indicated that the effects of lidocaine on the proliferation and apoptosis of Y79 cells were mediated by upregulating miR-520a-3p.

To investigate the effects of lidocaine, miR-520a-3p and EGFR on tumor growth in mice, Y79 cells transfected with miR-520a-3p inhibitor or NC were injected subcutaneously into mice. First, the level of miR-520a-3p and EGFR in RB model mice and the normal control mice was detected, and RB tissues from the RB model mice (Cancer tissues) and the normal control mice (Normal tissues) were collected. RT-qPCR and western blot analyses revealed that miR-520a-3p expression was significantly downregulated in RB tumor tissues from mice compared with normal tissues (Fig. 8A), whereas EGFR expression was upregulated (Fig. 8B and C). It was observed that transfection with inhibitor suppressed the expression of miR-520a-3p in tumor tissues and promoted the expression of EGFR compared with the control, whereas treatment with lidocaine induced opposing effects (Fig. 8D-F). Furthermore, compared with lidocaine treatment alone, transfection of Y79 cells with miR-520a-3p inhibitor significantly downregulated the expression of miR-520a-3p and upregulated the expression of EGFR in the resulting tumor tissues following treatment with lidocaine. These in vivo results were consistent with the findings of the in vitro experiments. Finally, the volume and weight of tumors were analyzed. It was demonstrated that transfection with inhibitor significantly increased tumor volume and weight compared with the control, whereas lidocaine treatment induced opposing effects (Fig. 8G and H). Furthermore, combining inhibitor transfection and lidocaine treatment significantly increased the volume and weight of tumors compared with lidocaine treatment alone.