MiR-613 suppresses retinoblastoma cell proliferation, invasion, and tumor formation by targeting E2F5

Abstract

Retinoblastoma is a common intraocular malignancy that occurs during childhood. MicroRNAs play critical roles in the regulation of retinoblastoma initiation and progression, and aberrant expression of miR-613 had been reported in various types of cancer. However, the role and mechanism of its function in retinoblastoma are still unclear. In this study, we found that miR-613 was downregulated in retinoblastoma tissues and cell lines. Overexpression of miR-613 suppressed retinoblastoma cell proliferation, migration, and invasion and induced cell cycle arrest in vitro. Additionally, overexpressed miR-613 also inhibited tumor formation of retinoblastoma cells in vivo. We further identified E2F5 as a direct target of miR-613. Reintroduction of E2F5 without 3′-untranslated region reversed the inhibitory effects of miR-613 on cell proliferation and invasion. Our data collectively indicate that miR-613 functions as a tumor suppressor in retinoblastoma through downregulating E2F5, supporting the targeting of the novel miR-613/E2F5 axis as a potentially effective therapeutic approach for retinoblastoma.

Introduction

Retinoblastoma (RB) is a common intraocular malignancy that occurs during childhood.¹ Most patients cannot be diagnosed during the early stage of RB, and delayed diagnosis is strongly associated with a high mortality rate, especially in developing countries.² RB extends along optic nerve into the brain and easily metastasizes.³ Currently, chemotherapy is commonly used to reduce the rate of death and to improve the prognosis of patients with RB.⁴ Therefore, looking for useful biomarkers and exploring novel targets for RB treatment are urgently demanding.

MicroRNAs (miRNAs) are a family of short, small, noncoding RNA molecules containing 19–25 nucleotides. The miRNAs could bind to the 3′-untranslated regions (3′-UTRs) of messenger RNAs (mRNAs), then promote the miRNA-mRNA-induced silencing complex formation, and degrade the targeted mRNAs.^4–7 In mammals, miRNAs regulate expression of more than half genes that are related to physiological processes, such as differentiation and metabolism, apoptosis, cell proliferation, and pathological processes, including development of neurological diseases, cardiovascular diseases, and tumors.^8–10 MiRNAs function as potent oncogenes and tumor-suppressor genes that play complex roles in cancer development. Accumulating data have shown that miRNAs are emerging candidates for new therapeutic targets and biomarkers with the ability to predict the prognosis of patients with tumors. Currently, multiple miRNAs have been identified as key regulators in RB, including miR-145,¹¹ miR-204,¹² miR-365b-3p,¹³ miR-31, and miR-200a.¹⁴ They may act as diagnostic biomarkers and/or therapeutic targets for RB. These findings suggest that targeting the miRNAs may potentially lead to a novel strategy of diagnose and therapy to RB.

Some studies have demonstrated that miR-613 was deregulated and functioned as a tumor suppressor in many cancers, including esophageal squamous cell carcinoma,¹⁵ ovarian cancer,¹⁶ and prostate cancer.¹⁷ However, miR-613 expression in human RB and its role in RB cells remain unclear. In this study, we found that the expression of miR-613 was downregulated in RB tissues and cell lines. MiR-613 suppressed RB cell proliferation, invasion, and tumor formation by directly targeting E2F5. Our data demonstrate that miR-613 is a tumor suppressor in RB by downregulating E2F5, providing the target of miR-613/E2F5 axis as a potentially therapeutic agent for RB.

Materials and methods

Cell culture

The human RB cell lines SO-RB50, Y79, and WERI-Rb-1 and normal retinal pigmented epithelium cell line ARPE-19 were used in all experiments. WERI-Rb-1 cell line was purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). CARPE-19, SO-RB50, and Y79 cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). All the cells were maintained in basic RPMI 1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies) and 1% penicillin/streptomycin (Life Technologies) at 37°C in a humidified atmosphere containing oxygen and 5% CO₂.

Patients and specimens

We obtained human RB samples from 45 patients and 7 normal retinas and a complete set of follow-up data. All of the 45 RB patients received enucleation or enucleation + chemotherapy ± radiation therapy in the Department of Ophthalmology, Daping Hospital, Third Military Medical University, between February 2005 and November 2010. Of the 45 RB patients, there were 25 females and 20 males. The age of the patients was 0–7 years, with an average age of 2.6 years. All 45 RB patients were confirmed histopathologically and staged based on the American Joint Commission for Cancer (AJCC) staging system. The last follow-up date was at the end of December 2014. Seven normal retinas were obtained from patients who had died of conditions other than ophthalmologic diseases in Daping Hospital, Third Military Medical University. We have written informed consent from the donor or family members. In this study, all human participants and human specimens were approved by the Ethics Committee of the Third Military Medical University, and informed consent was obtained from all patients.

Transient transfection

The miR-613 mimics, miR-613 inhibitor, and their corresponding negative controls were synthesized by RiboBio (Guangzhou, China). Transient cell transfection was performed using Lipofectamine 2000 (Invitrogen, Pittsburgh, PA, USA) according to the manufacturer’s instruction. Cells were collected 48 h after transfection. The plasmid expressing E2F5 was obtained as previously described.¹⁸ Viral packaging was performed according to standard protocols.

RNA extraction, reverse transcription, and quantitative real-time polymerase chain reaction

Total RNA was extracted from cultured cells using QIAzol and the miRNeasy Mini Kit (Qiagen, Dusseldorf, Germany) according to the manufacturer’s protocol. The purity and concentration of all RNA samples were quantified using NanoDrop 2000 (Thermo Fisher Scientific, Pittsburgh, PA, USA). The quantitative reverse transcription polymerase chain reaction (qRT-PCR) method has been previously described.¹⁹ qRT-PCR was performed in 96-well plates using StepOnePlus (Applied Biosystems, Pittsburgh, PA, USA). All reactions were performed in triplicate. Hsa-miR-613 and endogenous control RNЦ6B TaqMan miRNA assays were obtained from Applied Biosystems. SYBR Green qRT-PCR was performed for qRT-PCR of mRNA, and the b-actin housekeeping gene was used to normalize the variation in the complementary DNA (cDNA) levels. All reactions were carried out in triplicate, and all experiments were performed three times.

Cell proliferation and colony formation assays

Cell viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates at a density of 2 × 10³ cells per well and incubated for 1, 2, 3, 4, and 5 days. Next, 5 µL of MTT solution (5 mg/mL) was added to each well at different time points, and the reaction subsequently terminated with 200 µL dimethyl sulfoxide (DMSO). Absorbance was measured at 560 nm. All experiments were performed in triplicate. For the colony formation assay, cells were seeded in 6-well plates at a density of 500 cells per well and cultured for 14 days. Colonies were fixed with 30% formaldehyde and stained with 0.1% crystal violet. Colonies (containing > 50 cells) were counted under an optical microscope.

BrdU staining

For BrdU immunofluorescent staining, cells were grown on coverslips and incubated with 10 µg/mL BrdU (Sigma) for 30 min, then washed with phosphate-buffered saline (PBS), and fixed in 4% paraformaldehyde (PFA) for 20 min. Subsequently, cells were pre-treated with 1 mol/L HCl and blocked with 10% goat serum for 1 h, followed by a monoclonal rat primary antibody against BrdU (1:200, ab6326; Abcam, Cambridge, MA, USA) for 1 h and Alexa Fluor^® 594 goat anti-rat IgG secondary antibody (H + L; Invitrogen). 4′,6-Diamidino-2-phenylindole (DAPI; 300 nM) was used for nuclear staining; the percentage of BrdU was calculated at least from 10 microscopic fields (Nikon 80i; Nikon Corporation, Tokyo, Japan).

Cell migration and invasion

Migration and invasion were examined using a transwell chamber (Millipore, Billerica, MA, USA). For the migration assay, 1 × 10⁵ transfected cells were plated into the upper chamber and cultured in RPMI 1640, while RPMI 1640 with 10% FBS was added to the lower chamber. After 24 h incubation at 37°C, cells remaining on the upper surface of membrane were removed, and membrane was stained with 20% methanol and 0.5% crystal violet. Cell images were obtained using an inverted microscope (Olympus, Tokyo, Japan). For invasion assays, the upper chamber was precoated with Matrigel (BD Biosciences, San Jose, CA, USA).

Flow cytometry

For cell cycle analysis, 1 × 10⁶ cells were harvested and washed twice with cold PBS, followed by fixation with ice-cold 70% ethanol overnight at 4°C. After washing twice with PBS, the cells were incubated with propidium iodide (PI) (BD Biosciences, San Jose, CA, USA) and RNaseA for 30 min at room temperature. The cells were then analyzed using a FACS C6 (BD Biosciences, San Jose, CA, USA) with CellQuest software (D BioSciences, San Jose, CA, USA).

Western blot

Cells were lysed in a lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) with complete protease inhibitor cocktail (Roche, Basel, Switzerland), and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Cell lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to a polyvinylidene difluoride membrane. SDS-PAGE gels were calibrated using MagicMark XP Western Standard (Invitrogen). Then, membranes were incubated with a primary antibody against human E2F5 (1:1000; Abcam), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1000; Beyotime, Shanghai, China), CDK2 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CDK4 (1:500; Santa Cruz Biotechnology), Cyclin D1 (1:500; Santa Cruz Biotechnology), or Cyclin E (1:500; Abcam) at 4°C overnight. After three 10-min washes with tris-buffered saline with Tween 20 (TBST), the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature (1:1000; Santa Cruz Biotechnology). The targeted bands were visualized with enhanced chemiluminescence (ECL).

Tumor xenograft experiment

Female severe combined immunodeficiency (SCID) mice (4 weeks old) were purchased and housed in an specific-pathogen-free (SPF) room that was maintained at a constant temperature (22°C–25°C) and humidity (40%–50%). Tumor cells (1 × 10⁶) were injected subcutaneously into the flanks of nude mice following a previous protocol with minor modifications.²⁰ The tumor size was measured using a vernier caliper every week, and the volume was calculated with the following formula: V = (length × width²)/2. At the termination of the experiment, the tumor mass was harvested, weighed, and stored for immunostaining or protein extract. All studies were approved by the Institutional Animal Care and Use Committee of Tangdu Hospital.

Luciferase reporter assay

For the luciferase assay, Y79 cells were seeded in 96-well plates 24 h before transfection and cotransfected with the E2F5 wild-type (Wt) or mutant (Mt) 3′-UTR reporter vector, has-miR-613, or negative control (NC) and Renilla plasmid using Lipofectamine 2000 (Invitrogen). Luciferase activities were determined with the Dual-Luciferase Reporter System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Statistical analysis

All experiments were performed at least in triplicate and results were recorded. Statistical analysis was performed with the Statistical Package for the Social Sciences Version 16.0 software (SPSS, Chicago, IL, USA). The miR-613 in RB tissues and normal retina tissues was analyzed using t-test analysis. The clinicopathological features of RB patients that correlated with miR-613 low- and high-expression groups were determined by the chi-square test. The miR-613 expression in relation to overall survival (OS) rates was analyzed using the Kaplan–Meier analysis and the log-rank test. The Cox regression analysis was used for univariate analysis and multivariate analysis of OS. The p < 0.05 was considered statistically significant.

Results

Expression of miR-613 was downregulated in human RB tissues and cell lines

To examine miR-613 expression in RB, we detected the expression of miR-613 in a mass of RB tissues and cell lines by qRT-PCR. We first investigated miR-613 expression in primary RB and normal tissues; the median score of miR-613 was found to be 1.626 (interquartile range: 1.506–1.960) in normal tissues and 0.592 (interquartile range: 0.432–0.694) in primary RB. MiR-613 was downregulated in tumor samples, compared to the normal tissues (Figure 1(a)). Consistently, miR-613 was deregulated in tumor cell lines, compared with CARPE-19 and HRME1 (Figure 1(b)). To further examine clinical significance of miR-613 in RB, 45 patients were grouped into two groups based on the expression of miR-613 in RB tissues. We found that the expression of miR-613 was negatively correlated with TNM stage (p < 0.05), while there was no correlation with age, tumor size, and gender (Table 1). To determine whether alterations at the genetic locus of miR-613 could be implicated in RB patient prognosis, Kaplan–Meier analysis showed that RB patients with miR-613 low expression had shorter OS (p = 0.024) than those with miR-613 high expression (1.5-fold higher than adjacent tissue) (Figure 1(c)). These data suggested that the downregulation of miR-613 might play an important role in RB progression.

Figure 1.

Expression of miR-613 is downregulated in RB patients: (a) qRT-PCR analysis of miR-613 expression in human retinoblastoma cancer samples and normal tissues; miRNA abundance was normalized to U6 RNA. (b) qRT-PCR analysis of miR-613 expression in CARPE-19, HRME1, WERI-RB-1, and Y79 cells and (c) the Kaplan–Meier analysis of OS for retinoblastoma patients with the log-rank test.

All data are shown as the mean ± SD. All p values are based on the analysis of control versus treatment.

**p < 0.01

[Figure omitted. See PDF]

Table 1.

Correlation between clinicopathological features and expression of miR-613 in retinoblastoma patients.

Feature	All patients (n = 45, n (%))	Expression of miR-613		p value
		Downregulation	Upregulation
		31	14
Age at presentation (years)				0.642
<5	40 (88.8)	26	12
>5	5 (11.2)	5	2
Gender				0.393
Male	20 (42.2)	13	7
Female	25 (57.9)	18	7
Tumor enucleated location				1.000
Right	25 (55.6)	19	8
Left	20 (44.4)	12	6
cTNM classification
T classification				0.143
T1 + 2	25	13	12
T3 + 4	20	18	2
N classification				0.036*
N0	20	12	8
N1 + 2	25	19	6
cTNM stage				0.024*
I + II	16	9	7
III + IV	29	22	7
Largest tumor base (mm)				0.048*
<15	30 (66.6)	18	12
>15	15 (33.4)	13	2
Tumor thickness (mm)				0.782
<10	25 (55.6)	17	8
>10	20 (44.4)	14	6
Differentiation				0.031*
Well and moderate	26 (57.7)	15	11
Poor	19 (42.3)	16	3

*

p < 0.05.

MiR-613 inhibits cell proliferation in RB

To test the role of miR-613 in RB, the miRNA was overexpressed in Y79 and WERI-Rb-1 cells though transfection, and the MTT assay was employed to ascertain its effects on cell proliferation. Overexpression of miR-613 inhibited the proliferation of the two cell lines, compared with their controls (Figure 2(c) and (d)). In contrast, the growth of RB cells was increased after antagomiR-613 transfection compared to the cells transfected with antagomiR-NC (Figure 2(e) and (f)). Above data were confirmed by BrdU incorporation in the Y79 and WERI-Rb-1 cell lines, where the miR-613-overexpressing cells showed over a 40% reduction, while the miR-613-knockdown cells showed over a 70% increment in DNA synthesis compared to control cells in the two cell lines (Figure 2(g)). These data clearly suggested that miR-613 is a tumor suppressor in RB.

Figure 2.

MiR-613 suppresses RB cell proliferation: (a) qRT-PCR analysis of miR-613 expression in cells transfected with NC or miR-613 mimics; miRNA abundance was normalized to U6 RNA. (b) qRT-PCR analysis of miR-613 expression in cells transfected with NC or miR-613 inhibitor. MTT assay of (c) Y79 and (d) WERI-RB-1 cells transfected with miR-613-expressing or control mimics. MTT assay of (e) Y79 and (f) WERI-RB-1 cells transfection with miR-613-expressing or control inhibitor and (g) image and quantification of WERI-RB-1 and Y79 cells positive for BrdU staining.

All data are shown as the mean ± SD. All p values are based on the analysis of control versus treatment.

**p < 0.01

[Figure omitted. See PDF]

MiR-613 inhibited migration and invasion in RB cells

To investigate the role of miR-613 in metastasis of RB, migration and invasion assays were performed in Y79 and WERI-Rb-1 cells transfected with miR-613 mimic and NC by transwell assay. Our data demonstrated that overexpression of miR-613 significantly inhibited the migration (Figure 3(a) and (b)) and invasion (Figure 3(c) and (d)) of Y79 and WERI-Rb-1 cells, suggesting that miR-613 suppressed the metastasis of RB cells.

Figure 3.

MiR-613 inhibits migration and invasion in RB cells. (a and b) MiR-613 decreased migration ability of Y79 and WERI-RB-1 cells; cell migration ability was determined by transwell migration assay. (c and d) MiR-613 suppressed invasion ability of Y79 and WERI-RB-1 cells; cell invasion ability was determined by transwell invasion assay.

**p < 0.01

[Figure omitted. See PDF]

MiR-613 induces cell cycle arrest in RB cells

Since the cell cycle progression usually regulates cell proliferation, the Y79 cell cycle was analyzed by flow cytometry to examine whether miR-613 suppresses cell proliferation by inhibiting the cell cycle. The population in the G1 phase was significantly increased and cell population in the S phase was reduced after miR-613 mimics transfection (Figure 4(a) and (b)). However, transfection of miR-613 antagomir promoted the cell cycle progression of RB cells (Figure 4(c) and (d)). Western blot assay was obtained from the detection of cyclin D1, cyclin E, CDK2, and CDK4 expression. We found that the expressions of cyclin E and CDK2 were reduced in the miR-613-treated cells, whereas transfection of miR-613 antagomir increased the expressions of CDK2 and cyclin E (Figure 4(e) and (f)). Our results indicate that miR-613 induces cell cycle arrest at G1 phase in RB cells.

Figure 4.

MiR-613 suppresses the cell cycle progression of RB cells by inhibiting cyclin E and CDK2. After (a and b) miR-613 mimics or (c and d) inhibitor were transfected, the distribution of cell cycle was detected by flow cytometry. The experiments were repeated three times with similar results and representative graphs were illustrated. (e and f) Expression of cyclin D1, cyclin E, CDK2, and CDK4 were examined by western blot.

*p < 0.05

[Figure omitted. See PDF]

MiR-613 impairs colony formation and tumor formation of RB cells in immunodeficient mice

To further assess the effects of miR-613 expression in colony formation, soft agar assay was employed in vitro. As shown in Figure 5(a), the colonies were smaller and lesser in miR-613 mimics–treated cells compared with the controls (Figure 5(a) and (b)). To examine the effect of miR-613 on tumor growth in vivo, stable overexpressing miR-613 or NC Y79 cells were injected into the flank of nude mice. The tumor growth was closely monitored. The mice were sacrificed after 35 days. Tumor growth of the miR-613-overexpressing group was significantly slower than the control group (Figure 5(c)). Additionally, the average tumor weight and volume of the miR-613-overexpressing group was lower than their controls (Figure 5(d) and (e)). To determine whether miR-613 inhibited the tumor progression of RB cells by decreasing cell proliferation, the well-known proliferation marker Ki67 was examined in the tumor tissues by immunohistochemical (IHC) staining. As shown in Figure 5(f), the expression of Ki67 in tumor tissues formed by the miR-613-overexpressing Y79 cells was decreased. The results suggested that miR-613 most likely suppressed the tumor progression of RB cells by inhibiting cell proliferation.

Figure 5.

Overexpression of miR-613 inhibits tumorigenicity. (a and b) Representative images and quantification of the colony formation on Y79 cells transfected with miR-613-expressing or control mimics. (c) Representative tumor tissues extracted from mice inoculated with Y79 cells expressing miR-613 or NC. (d) Measurement of tumor volumes at the indicated time points. (e) Calculated tumor weights after 35 days. (f) Immunohistochemical staining for Ki67 in tumor tissues.

All data are shown as the mean ± SD. All p values are based on the analysis of control versus treatment.

**p < 0.01

[Figure omitted. See PDF]

E2F5 was a direct target gene of miR-613 in RB cell

Bioinformatics assay with TargetScan7.0 algorithms predicted E2F5 as a hypothetical target gene of miR-613 (Figure 6(a)). Co-transfected with miR-613 mimic or NC and the E2F5 3′-UTR reporter plasmid in Y79 cells, the relative luciferase activity of the reporter was inhibited by miR-613 mimic; however, there is no changes in the luciferase activity of MT reporter (Figure 6(b)). Quantitative RT-PCR and western blot assays were employed to detect the effects of miR-613 on the expression of E2F5. We found that transfection with miR-613 mimics significantly decreased the mRNA and protein level of E2F5 in Y79 cells. In contrast, antagomiR-613 increased E2F5 expression in the cell line (Figure 6(c) and (d)).

Figure 6.

MiR-613 directly targets the E2F5 3′-UTR (a) wild-type and mutant miR-613 target sequences of the E2F5 3′-UTR. (b) Relative luciferase activity of Y79 cells after cotransfection with wild-type (Wt) or mutant (Mt) E2F5 3′-UTR reporter genes along with miR-613 or NC. (c) qRT-PCR analysis and (d) western blot of E2F52 expression in the indicated cells.

All data are shown as the mean ± SD. All p values are based on the analysis of control versus treatment.

**p < 0.01

[Figure omitted. See PDF]

E2F5 rescued the effect of miR-613-mediated RB cell proliferation, migration, and invasion

To determine whether E2F5 is a functional target of miR-613, we employed gain-of-function analyses by transfecting E2F5 plasmids without 3′-UTR into miR-613-overexpressing Y79 cells (Figure 7(a)). MTT analysis demonstrated that E2F5 overexpression promoted the miR-613-overexpressing Y79 cell proliferation (Figure 7(b)). Migration and invasion analysis demonstrated that overexpression of E2F5 increased the ability of migration and invasion in miR-613-overexpressing Y79 cells (Figure 6(c) and (d)).

Figure 7.

E2F5 rescued the effect of miR-613-inhibited RB cell proliferation, migration, and invasion. (a) The protein expression of E2F5 was measured by western blot. (b) The cell proliferation was measured by MTT analysis. (c and d) Migration and invasion assays demonstrated that ectopic expression of E2F5 promoted the miR-613-overexpressing Y79 cell migration and invasion.

All data are shown as the mean ± SD. All p values are based on the analysis of control versus treatment.

**p < 0.01

[Figure omitted. See PDF]

Discussion

MiRNAs have been identified as putative oncogenes or cancer suppressors in various cancers. Recent studies have demonstrated that multiple miRNAs play important roles in RB. MicroRNA-613 is a family of miRNAs that are strongly associated with cancer development. Specifically, accumulating evidence suggests that miR-613 plays the role as a tumor suppressor in many cancers including esophageal squamous cell carcinoma, ovarian cancer, and prostate cancer,^15–17 suggesting that miR-613 might exert important effects on RB. However, the function and the potential mechanism of miR-613 in RB have not been explored.

MiR-613 has been found to be underexpressed in ovarian cancer, esophageal squamous cell carcinoma, and prostate cancer, suggesting its role as a tumor suppressor in these cancers. For example, Guan et al.¹⁵ showed that miR-613 is significantly reduced in esophageal squamous cell carcinoma (ESCC) patients. Fu et al.¹⁶ found that the expression of miR-613 was downregulated in ovarian tissues and overexpression of miR-613 inhibited ovarian cancer cell proliferation and invasion through targeting KRAS expression. Ren et al.¹⁷ showed that miR-613 inhibition promoted cell proliferation and epithelial–mesenchymal transition (EMT) by decreasing Frizzled7 expression in prostate cancer. However, the function of miR-613 in RB remains still unknown. In this study, we first detected miR-613 expression in the RB tissues. Our results demonstrated that the expression of miR-613 was downregulated in RB tissues compared to the normal issues. Moreover, overexpression of miR-613 inhibited cell proliferation, migration, invasion, and tumor formation in RB.

It is well known that miRNAs play its biological function through regulating their target genes.²¹ Therefore, we searched the target gene of miR-613 through the TargetScan7.0 bioinformatics algorithm. E2F5 was picked out as a potential target of miR-613 contains a target sequence at position 542–549 of the E2F5-3′-UTR. The dual-luciferase reporter assay confirmed that E2F5 is a direct target gene of miR-613. In addition, the mRNA and protein levels of E2F5 were significantly decreased in miR-613-overexpressing Y79 cells compared with their controls. E2F5, a member of the E2 promoter binding factor (E2F) family, which is a transcription factor, participated in the regulation of cellular proliferation.^22–24 Accumulating evidence suggested that E2F5 played important roles in cancer development, including hepatocellular carcinoma, breast, ovarian, colon, osteogenic sarcoma, and esophageal squamous cell carcinoma.^25–29 E2F5 was upregulated and involved in cell growth and proliferation through regulating cell cycle genes in RB.³⁰ In this study, we found that E2F5 is a direct target gene of miR-613 in RB cells. Overexpression of E2F5 rescued the effect of miR-613-mediated RB cell proliferation, migration, and invasion. Our data suggested that miR-613 was a tumor suppressor via the post-transcriptional regulation of E2F5.

In conclusion, the expression of miR-613 is downregulated in RB patients and cell lines. Our data indicate that miR-613 negatively regulates the expression of E2F5 in RB cells by binding to the 3′-UTR of E2F5 mRNA. MiR-613 suppresses RB cell proliferation, cell cycle progression, invasion, and tumor growth by directly targeting E2F5, supporting this miRNA as a potential therapeutic agent for RB.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical standards

Prior informed consent was obtained from all patients and the study was approved by the Ethics Committee of Nanjing University.

Funding

This study was supported by a grant from the Natural Science Foundation of Jiangsu Province (BK2012777) and National Natural Science Foundation of China (NSFC; no. 81270979).