Lidocaine inhibits the progression of retinoblastoma in vitro and in vivo by modulating the miR‑520a‑3p/EGFR axis.

Retinoblastoma (RB) is a common malignant tumor in children. Lidocaine is a local anesthetic and anti‑arrhythmic drug, and has been reported to possess anti‑tumor properties. MicroRNAs (miRs) are a group of endogenous small noncoding RNAs that have important roles in various biological processes via actions on target genes. The aim of the present study was to investigate the effect of lidocaine on retinoblastoma in vitro and in vivo. CCK‑8 assay and flow cytometry assay were used to measure cell viability and apoptosis. The relationship between miR‑520a‑3p and EGFR was predicted and confirmed by TargetScan and dual‑luciferase reporter assay. For in vivo study, tumor xenograft was performed. In addition, gene and protein expression was detected using reverse transcription‑quantitative polymerase chain reaction and western blotting respectively. In the present study, it was observed that lidocaine inhibited the proliferation and induced the apoptosis of RB cells. miR‑520a‑3p was reported to be downregulated in RB tissues and cell lines; treatment with lidocaine increased the expression of miR‑520a‑3p in RB cells. The human epidermal growth factor receptor (EGFR) was identified as a direct target of miR‑520a‑3p, and its expression was negatively associated with that of miR‑520a‑3p. Additionally, EGFR was upregulated in RB tissues and cell lines; treatment with lidocaine decreased the expression of EGFR in RB cells. Furthermore, compared with treatment with lidocaine alone, the combination of transfection with miR‑520a‑3p inhibitor and lidocaine treatment significantly decreased the expression of miR‑520a‑3p, increased EGFR expression, promoted RB cell proliferation and reduced the apoptosis of cells in vitro, and increased tumor volume and weight in vivo. The results indicated that lidocaine reduced the proliferation and induced the apoptosis of RB cells by decreasing EGFR expression via the upregulation of miR‑520a‑3p, suggesting that the miR‑520a‑3p/EGFR axis may be a novel therapeutic target in the treatment of RB.

Introduction

Retinoblastoma (RB) is a malignant tumor derived from photoreceptor precursor cells (1). RB is frequently reported in children of <3 years old, and is the most common intraocular malignant tumor in infants and young children; however, it is rarely observed in adults (1,2). Familial RB is inherited via the transmission of mutations in Rb1, a tumor suppressor gene located at 13q14 (3).

Lidocaine, a derivative of cocaine, is a local anesthetic and anti-arrhythmic drug (4). Lidocaine is used in clinical settings for local skin and mucosal anesthesia, and regional nerve block, with anti-inflammatory and anti-bacterial effects (5,6). Increasing evidence has suggested that certain anesthetics have effects on tumor metastasis and recurrence (7,8); however, the effects of lidocaine on RB remain unknown.

MicroRNAs (miRNAs/miRs) are small noncoding RNAs (20–22 nucleotides in length); miRNAs have been associated with various types of cancer (9–12). miRNAs suppress the expression of genes by binding to the 3′-untranslated region (3′-UTR) of target mRNAs (13–15). It was recently reported that miR-520g was downregulated in esophageal cancer (16). Additionally, the miR-520 family is reported to have tumor suppressor roles in various human tumors, including breast (16) and renal and hepatocellular carcinoma (17,18). Previous studies have reported that miR-520a-3p was associated with the occurrence and development of numerous tumor types, such as colorectal and non-small cell lung cancer (19,20). Therefore, the expression of miR-520a-3p may have clinical value.

Abnormal expression of human epidermal growth factor receptor (EGFR) has been reported in various solid tumors (21,22). EGFR may regulate the proliferation and apoptosis of tumor cells, and has been identified as a therapeutic target in numerous human cancers (23,24). Lidocaine has been reported to inhibit the proliferation of human tongue cancer cells by inhibiting the activity of EGFR (25). Additionally, lidocaine promoted the apoptosis of colorectal cancer cells (26). Furthermore, miR-520a-3p inhibited the proliferation and promoted the apoptosis of colorectal cancer cells by targeting EGFR (27). Therefore, the study investigated the effects of lidocaine on the expression of miR-520a-3p and EGFR, and the proliferation and apoptosis of RB cells.

Materials and methods

Clinical specimens

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.

Cell culture and transfection

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.

Cell Counting Kit-8 (CCK-8) assay

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.

Flow cytometry assay

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.

Dual-luciferase reporter assay

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.

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

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.

Western blot assay

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.

Tumor xenograft experiment

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.

Statistical analysis

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.

Results

Effects of lidocaine on the proliferation and apoptosis of RB cells

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).

Figure 1.

Lidocaine inhibits the proliferation of retinoblastoma cells. Y79 cells were treated with control or lidocaine (50, 100, 500 or 1,000 µM). Cell Counting Kit-8 assays were performed at 0, 12, 24 and 48 h to determine the proliferation of Y79 cells. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. *P<0.05, **P<0.01 vs. control.

Figure 2.

Lidocaine induces the apoptosis of retinoblastoma cells. Y79 cells were treated with control or lidocaine (500 and 1,000 µM). Flow cytometry was performed at 24 and 48 h to determine the apoptosis of cells. Early apoptosis (Q4) and late apoptosis (Q2) were calculated and the apoptosis rate was presented. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. **P<0.01 vs. control. FITC, fluorescein isothiocyanate; PI, propidium iodide.

Effects of lidocaine on the expression of miR-520a-3p

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).

Figure 3.

Lidocaine upregulates the expression of miR-520a-3p in RB cells. (A) Expression of miR-520a-3p in RB and normal adjacent tissues. n=30/group. Data are presented as the mean ± standard deviation. **P<0.01 vs. normal tissues. (B) Expression of miR-520a-3p in RB cell lines and the retinal pigment epithelial cell line ARPE-19. *P<0.05, **P<0.01 vs. ARPE-19. (C) Expression of miR-520a-3p in Y79 cells following treatment with 500 µM lidocaine for 24 h. **P<0.01 vs. control. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. miR-520a-3p, microRNA-520a-3p; RB, retinoblastoma.

EGFR is a direct target gene of miR-520a-3p

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).

Figure 4.

EGFR is a direct target gene of miR-520a-3p. (A) TargetScan prediction of a binding site for miR-520a-3p in the EGFR 3′-UTR. (B) Luciferase activity in Y79 following co-transfection with mimic or NC and luciferase reporter plasmids containing WT-EGFR or MUT-EGFR. **P<0.01 vs. NC. Relative expression of (C) miR-520a-3p, and (D) EGFR mRNA and (E) protein in Y79 cells following transfection with NC or mimics. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. **P<0.01 vs. control. EGFR, epidermal growth factor receptor; 3′-UTR, 3′-untranslated region; miR-520a-3p, microRNA-520a-30; MUT-EGFR, mutated EGFR 3′-UTR; WT-EGFR, wild-type EGFR 3′-UTR; NC, negative control; mimic, miR-520a-3p mimic.

Effects of lidocaine on EGFR expression

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).

Figure 5.

Lidocaine downregulates the expression of EGFR in RB cells. Expression of EGFR (A) mRNA and (B) protein in RB and normal adjacent tissues (a1, a2 and a3, RB tissues; b1, b2 and b3, normal adjacent tissues) n=30/group. Data are presented as the mean ± standard deviation. **P<0.01 vs. normal tissues. Expression of EGFR (C) mRNA and (D) protein in RB cell lines and the retinal pigment epithelial cell line ARPE-19 and RB cell lines. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. *P<0.05, **P<0.01 vs. ARPE-19. Expression of EGFR (E) mRNA and (F) protein in Y79 cells following treatment with 500 µM lidocaine for 24 h. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. **P<0.01 vs. control. RB, retinoblastoma; EGFR, epidermal growth factor receptor.

Effects of lidocaine on RB cells are mediated via upregulation of miR-520a-3p

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.

Figure 6.

Effects of lidocaine on the expression of miR-520a-3p and EGFR in retinoblastoma cells. Y79 cells were transfected with NC or inhibitor for 24 h, and then treated with lidocaine or control for 24 h. Expression of (A) miR-520a-3p, and (B) EGFR mRNA and (C) protein in the various groups. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. **P<0.01 vs. control; &&P<0.01 vs. 500 µM lidocaine. miR-520a-3p, microRNA-520a-3p; NC, negative control; inhibitor, miR-520a-3p inhibitor; EGFR, epidermal growth factor receptor.

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.

Figure 7.

Lidocaine inhibits the proliferation and promotes the apoptosis of retinoblastoma cells via upregulation of miR-520a-3p. Y79 cells were transfected with NC or inhibitor for 24 h, and then treated with lidocaine or control for 24 h. (A) Proliferation of Y79 cells as determined by a Cell Counting Kit-8 assay. (B) Apoptosis of Y79 cells as determined by a flow cytometry assay. (C) Early apoptosis Early apoptosis (Q4) and late apoptosis (Q2) were calculated and the apoptosis rate was presented. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. **P<0.01 vs. control; &&P<0.01 vs. 500 µM lidocaine. NC, negative control; miR-520a-3p, microRNA-520a-3p; inhibitor, miR-520a-3p inhibitor; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Effects of lidocaine on the volume, weight and expression of miR-520a-3p/EGFR in vivo

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.

Figure 8.

Lidocaine reduces the volume and weight of RB tumors in vivo via upregulation of miR-520a-3p. First, the level of miR-520a-3p and EGFR in RB model mice and normal control mice was detected, and RB tissues from the RB model mice (Cancer tissues) and the normal control mice (Normal tissues) were collected. Expression of (A) miR-520a-3p, and (B) EGFR mRNA and (C) protein in RB and adjacent tissues of mice. **P<0.01 vs. normal tissues. Tumor xenografts were implanted in mice via subcutaneous injection of 2×106 Y79 cells suspended in 100 µl serum-free RPMI-1640 medium on the sides of the posterior flank of nude mice. At 18 days after tumor inoculation, mice were subsequently treated with lidocaine (1.5 mg/kg) via tail vein injection. Y79 cells were transfected with NC or inhibitor prior to subcutaneous injection. Expression of (D) miR-520a-3p, and (E) EGFR mRNA and (F) protein in tumor tissues 18 days after tumor inoculation. (G) Tumor volume and (H) weight in the various groups. Data are presented as the mean ± standard deviation of three independent experiments performed in triplicate. *P<0.05, **P<0.01 vs. control; &P<0.05, &&P<0.01 vs. lidocaine. RB, retinoblastoma; miR-520a-3p, microRNA-520a-3p; EGFR, epidermal growth factor receptor; inhibitor, miR-520a-3p inhibitor; NC, negative control.

Discussion

The abnormal expression of various miRNAs has been associated with the onset and development of cancer (31–34). miRNAs may serve oncogenic or tumor suppressor roles in the development of tumors (35,36); previous studies have reported that whether an miRNA is an oncogene or a tumor suppressor is dependent upon the type of cancer (35,36).

The abnormal expression of members of the miR-520 family has been reported in a variety of tumors and transcripts of the miR-520 family are regarded as important regulators of oncogenes (16). The miR-520 family was downregulated in colon cancer (19). Additionally, previous studies reported that miR-520d-3p was identified as a tumor suppressor gene in ovarian cancer (37) and gastric cancer (38). A recent study demonstrated that miR-520g suppresses the proliferation and promotes the apoptosis of esophageal squamous cell carcinoma cells by targeting cysteine rich C-terminal 1 (15). Lidocaine is an amide local anesthetic that promotes the apoptosis of colorectal cancer cells (26,27). In a previous study, lidocaine suppressed the proliferation of human tongue cancer cells by reducing the activity of EGFR (25). It has been reported that EGFR is a target of miR-520a-3p, and miR-520a-3p inhibits the proliferation and induces the apoptosis of colorectal cancer cells by targeting EGFR (19). The aim of the present study was to investigate whether miR-520a-3p inhibits the proliferation of RB cells by targeting EGFR.

Previous studies demonstrated that anesthetics such as morphine or propofol affected the malignancy of solid tumors (39,40). Lidocaine has been hypothesized to inhibit the invasive ability of tumor cells at concentrations used during surgical procedures (41). In the present study, it was revealed that lidocaine suppressed the proliferation and promoted the apoptosis of RB in vitro. The findings indicated that lidocaine promoted the expression of miR-520a-3p. miR-520a-3p is a potential tumor suppressor that has been associated with human cancers such as non-small cell lung cancer (42). Li et al (43) revealed that miR-520a-3p suppressed the development of breast cancer by targeting cyclin D1 and cluster of differentiation 44. Zhang et al (19) revealed that miR-520a-3p reduced cell migration, promoted cell apoptosis and arrested the cell cycle of colorectal cancer cells at the G0/G1 phase, and decreased tumor growth in xenograft mice, potentially by targeting EGFR.

In the present study, it was observed that lidocaine inhibited the proliferation and promoted the apoptosis of Y79 cells by upregulating miR-520a-3p. Furthermore, it was revealed that EGFR was highly expressed in RB cells and tissues; lidocaine treatment downregulated EGFR expression at the mRNA and protein levels. Tumor xenograft experiments demonstrated that lidocaine suppressed the growth of RB xenograft tumors in vivo. These effects were eliminated following transfection of RB cells with inhibitor, indicating that the effects of lidocaine were mediated via regulation of miR-520a-3p. Conversely, the effects of lidocaine injection directly into tumor tissue were not investigated, presenting a potential limitation of the present study.

In conclusion, the findings of the present study indicated that lidocaine suppresses the development of RB in vitro and in vivo by upregulating miR-520a-3p, with EGFR the potential downstream target. Therefore, lidocaine may serve as a potential therapeutic agent, whereas the miR-520a-3p/EGFR axis may be a novel target in the treatment of RB.