MiR-486-3p inhibits the proliferation, migration and invasion of retinoblastoma cells by targeting ECM1

Retinoblastoma (RB) is a common intraocular tumor in infants and young children, which seriously endangers the vision and life of children [1,2]. Early and timely treatment of RB patients can achieve better survival rate and retention of visual function [3,4], while the tumor will grow and spread rapidly within the eyeball, invade the vitreous body and subretinal space, spread to the skull along the optic nerve, leading to the death of the patient, without timely diagnosis or treatment [5–7]. At present, the common treatment methods for RB are transpupillary thermal therapy, cryotherapy, chemotherapy, and ophthalmectomy, orbital exenteration and gene therapy [8,9]. Gene therapy is the treatment of a disease by manipulating a therapeutic gene or disease-related gene as a therapeutic target, which has a good prospect in the application of multiple tumor therapies [10,11]. Thus, it is important to explore the biological and molecular mechanisms of RB in order to identify new diagnostic criteria and therapeutic targets.

MicroRNAs (miRNAs) are non-coding single-stranded RNAs that inhibit or degrade the mRNA by binging with the target mRNA 3′-untranslation region (3′-UTR), and regulate the target genes expression [12–14]. Previous studies have demonstrated that miRNA expression may be specific to certain types of cancer and tumor-derived miRNAs may be stably detected in the plasma or serum [15,16]. In recent years, it has been reported that plasma miR-320, miR-let-7e and miR-21 acted as novel potential biomarkers for the detection of RB [17]. In addition, studies have found that many miRNAs are abnormally expressed in RB, such as miR-192, miR-34a and miR-376a [18–21]. miR-125b promoted tumor growth and suppressed apoptosis by targeting DRAM2 in RB [22]. miR-188-5p promoted epithelial–mesenchymal transition via targeting DNA binding 4 through Wnt/β–catenin signaling in RB [23]. These studies indicate that abnormal miRNA expressed may have some influence on the development of RB. Therefore, the discovery of abnormally expressed miRNAs and their target genes in RB may provide a new idea for the research and treatment of the molecular mechanism of RB.

Studies have shown that miR-486-3p is abnormally expressed in a variety of cancer cells, such as oral cancer, cervical cancer and oral tongue squamous cell carcinoma [24–26]. The down-regulation of miR-486-3p is considered as one of the causes for metastasis of cervical cancer [25]. Venkatesan et al. [27] found that miR-486-3p expression was decreased in the RB tumor tissues, however, the role of miR-486-3p in RB is unclear. In the present study, clone formation assay, flow cytometry, transwell chamber and other experimental methods were used to investigate the effect of overexpression of miR-486-3p on the proliferation, apoptosis and invasion ability of RB, in order to provide new ideas for clinical treatment of RB.

RPMI-1640 medium (31870082; Gibco, U.S.A.); 10% fetal bovine serum (FBS, 16140071; Gibco, U.S.A.); 100 U/ml penicillin (10378016; Gibco, U.S.A.); 100 μg/ml streptomycin (10378016; Gibco, U.S.A.); Lipofectamine 2000 Transfection Reagent (11668030; Invitrogen, U.S.A.); Wright’s–Giemsa’s staining solution (G5225; GBCBIO technologies Inc., China); paraformaldehyde (P0099-500ml; Beyotime, China); Crystal Violet (C0121; Beyotime, China); TRIzol reagent (15596026; Invitrogen, U.S.A.); SYBR miRNA detection assays (Takara, China); RIPA buffer (P0013K; Beyotime, China); PVDF membrane (Merck, Germany); Cell counting kit-8 (CCK-8, C0037; Beyotime, China); Annexin V-FITC Apoptosis Detection kit (APOAF; Sigma–Aldrich, U.S.A.); PrimeScript RT Master Mix kit (RR036A; Takara, China); BCA protein assay kit (23225; Pierce, Germany).

Human tissue samples (including RB tumor tissues and normal retinal tissues) of 45 patients with RB who were underwent eye enucleation in Shengjing Hospital of China Medical University were collected. All patients were not treated with radiotherapy or chemotherapy before operation, the postoperative pathological sections were diagnosed as RB. The samples were frozen and stored in liquid nitrogen, the tissues used for pathological examination were fixed by 4% paraformaldehyde. The experimental procedure was approved by Ethics Committee of Shengjing Hospital of China Medical University, and the written informed consent of each patient was obtained.

Human retinal pigment epithelial cells (ARPE-19) and RB cell lines (Y79, SO-RB50, WERI-Rb-1) were both purchased from the Typical Cell Culture Collection Committee of the Chinese Academy of Sciences Library (http://www.cellbank.org.cn/search.asp?a=1). All cells were cultured in RPMI-1640 medium containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C with 5% CO₂.

Transfection of miRNA: Y79 and SO-RB50 were transfected with miR-486-3p mimic (mimic group) or miR-486-3p mimic control (mimic control group) by Lipofectamine 2000 Transfection Reagent, according to the manufacturer’s instructions. The single cell suspension at a density of 5 × 10⁴ cells/ml was prepared and seeded into the 96-well plate. The miRNA was mixed with OPTIM-MEM 1, and then the Lipofectamine 2000 reagent and OPTIM-MEM 1 were added and incubated for 20 min. Then, the mixture was incubated at 37°C with 5% CO₂ for 48 h. The untreated cells were set up as control group (blank group).

The expression vector with wild-type/mutant ECM1-3′-UTR sequences was designed and synthesized by Shanghai GenePharma Co., Ltd (China). Y79 and SO-RB50 were co-transfected with miR-486-3p mimic and ECM1-WT or ECM1-MUT. The untreated cells were set up as control group (blank group). The experiment was divided into four groups: blank-ECM1-WT group, mimic-ECM1-WT group, blank-ECM1-MUT group and mimic-ECM1-MUT group. For another transfection experiments, Y79 and SO-RB50 were co-transfected with miR-486-3p mimic control or miR-486-3p mimic and ECM1-WT (ECM1) or empty vector (negative control, NC). The experiment was divided into four groups: Mimic control+NC group, Mimic control+ECM1 group, Mimic+NC group, and Mimic+ECM1 group. The full list of the primers used for transfection are shown in Table 1.

CCK-8 kit was performed to determine the cell viability of Y79 and SO-RB50. The cell density was adjusted to 5 × 10⁵ cells/ml and inoculated in a 96-well plate and cultured for 24, 48, 72 h, then the CCK-8 solution was added and again cultured for 2 h. Optical density (OD) of each well was measured at the wavelength of 450 nm by a microplate (Model 680, Bio-Rad, U.S.A.).

Cells were re-suspended in RPMI-1640 and seeded into a 35-mm culture dish with 500 cells per plate, and cultured at 37°C in a 5% CO₂ cell culture incubator. After 14 days, the cells were fixed with 4% poly-methanol for 20 min, then stained with Wright’s–Giemsa’s staining solution for 10 min. After natural air drying, the images were observed and counted under the inverted phase contrast microscope (CK40, Olympus, Japan).

The Annexin V-FITC Apoptosis Detection kit was performed to determine cell apoptosis. Cells at the density of 1 × 10⁶ cells/ml were resuspended in binding buffer. Then, the mixture was mixed with Annexin V-FITC and incubated in dark environment for 10 min. The propidium iodide (PI) was added, and then incubated in dark environment for 15 min. Cell apoptosis was analyzed by flow cytometry (BD FACSCalibur, BD Biosciences, U.S.A.).

Cells were seeded into a 24-well plate at a density of 5 × 10⁵ cells/ml, and a straight wound was created by a sterile pipette tip. At 0 and 48 h after the wounding, the cells were imaged by inverted phase-contrast microscope (Eclipse TS100, Nikon, Japan) and the cell migration was analyzed by NIS-Element Basic Research v3.2 software (Nikon, Japan).

Transwell chambers (8-μm pores, Corning Company, U.S.A.) pre-coated with Matrigel were placed into a 24-well plate, and the cells were seeded on the upper champers at a density of 2 × 10⁵ cells/well. The serum-free medium was added to the upper chamber, the RPMI-1640 medium with 10% FBS was added to the lower chamber. After 48 h, the excess culture fluid was removed and the invaded cells were fixed with 4% paraformaldehyde for 5 min, and then stained with 0.1% Crystal Violet for 5 min. The image was observed under an inverted microscope (TS100, Nikon, Japan).

The TargetScan 7.2 (http://www.targetscan.org/) was used to predict the potential target of miR-486-3p. The miR-486-3p mimic and ECM1-WT or ECM1-MUT were co-transfected into Y79 and SO-RB50 by Lipofectamine 2000 Transfection Reagent, respectively. The untreated cells were set up as control group (blank group). The luciferase activities were measured using the Dual-luciferase Reporter Assay kit (Promega, U.S.A.).

Total RNA was isolated from cells by TRIzol reagent. RNA purity and concentration were determined by NanoDrop 2000 (Thermo Fisher Scientific, U.S.A.). The total RNA (2 μg) was reverse transcribed to cDNA by PrimeScript RT Master Mix kit. Then, SYBR miRNA detection assays and Opticon real-time PCR Detection System (ABI 7500, Life Technology, U.S.A.) were used for quantitative analysis. The response procedure: 94°C for 5 min, (94°C for 30 s, 58°C for 30 s, 72°C for 30 s) ×35 cycles, and 72°C for 5 min. The expression level was calculated using 2^−ΔΔC_T method. U6 was used as internal reference gene, and the primers for quantitative real-time polymerase chain reaction (qRT-PCR) used in the present study were shown in Table 2.

Total protein was lysed with RIPA buffer and then was quantified with BCA protein assay kit. The protein (20 μg) was separated by 10% SDS/PAGE, and transferred to PVDF membrane. The membrane was blocked with 5% skim milk solution for 1 h, and then incubated with primary antibodies overnight at 4°C: anti-Bcl-2 (ab59348, 1:1000, Acbam), anti-Bax (ab32503, 1:1000, Acbam), anti-Cleaved Caspase-3 (C caspase-3, #9661, 1:1000, CST), anti-GAPDH (ab8245, 1:2000, Acbam), anti-extracellular matrix protein 1 (ECM1, ab126629, 1:1000, Acbam). Next, the membrane was incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies for 2 h: goat anti-rabbit IgG H&L (HRP) (ab205718, 1:2000, Abcam), goat anti-mouse IgG H&L (HRP) (ab205719, 1:2000, Abcam). GAPDH was used as a control. The protein band was analyzed by Pierce Western Blotting ECL substrate kit (Thermo Fisher Scientific, U.S.A.) and BandScan 5.0 system (Bio-Rad, Hercules, U.S.A.).

Experiments were repeated at least three times. SPSS 20.0 system (SPSS Inc., U.S.A.) was used for statistical analysis. All experimental data was presented as the mean ± standard deviation (SD). Comparisons between groups were analyzed with Student’s t tests or one-way ANOVA. P<0.05 was considered to be statistically significant.

The expression of miR-486-3p in tissues and cell lines (ARPE-19, Y79, SO-RB50, WERI-Rb-1) was measured by qRT-PCR. The miR-486-3p level was lower in RB tumor tissues (RB group) than that in normal retinal tissues (normal group, Figure 1A, P<0.05). Moreover, compared with ARPE-19, the miR-486-3p level was lowly expressed in Y79, SO-RB50 and WERI-Rb-1 (Figure 1B, P<0.05). Compared with normal tissues and cells, the expression of miR-486-3p was significantly decreased in RB tissues and cells, which may indicate that miR-486-3p was involved in the occurrence and development of RB.

Our previous experiments showed that miR-486-3p level was lower in Y79 and SO-RB50; therefore, Y79 and SO-RB50 were selected to investigate the effect of miR-486-3p on RB. Y79 and SO-RB50 were transfected with miR-486-3p mimic control (mimic control group) and miR-486-3p mimic (mimic group), respectively, and a blank group was set. The transfection of overexpressed miR-486-3p was detected by qRT-PCR, and miR-486-3p level was highly expressed in mimic group (including Y79 and SO-RB50) (Figure 2A, P<0.05), miR-486-3p mimic up-regulated miR-486-3p expression.

Cell viability and the ability of cell cloning were detected by CCK-8 and clone formation assay. As shown in Figure 2B,C, in Y79 and SO-RB50 cell lines, the cell viability was lower in mimic group than that in blank group and mimic control group (P<0.05). Clone formation assay demonstrated that the clones in Y79-mimic and SO-RB50-mimic groups were lower than those in corresponding blank and mimic control groups (Figure 2D,E, P<0.05). The cell viability and the ability of cell cloning were inhibited by miR-486-3p.

For the apoptosis of Y79 and SO-RB50, the apoptosis rate and the expressions of related apoptotic proteins were detected by flow cytometry and Western blot. In Y79 cell line, the apoptosis rate was higher in mimic group than that in blank and mimic control groups (Figure 2F,G, P<0.05). Moreover, compared with blank and mimic control groups, in mimic group, Bcl-2 expression was down-regulated, while the Bax and C caspase-3 expressions were up-regulated (Figure 3A,B, P<0.05). In addition, in SO-RB50 cell line, compared with blank and mimic control groups, the apoptosis rate (Figure 2F,G) and the Bax and C caspase-3 expressions (Figure 3C,D) in mimic group were up-regulated, and the Bcl-2 expression (Figure 3C,D) was down-regulated (P<0.05). We found that miR-486-3p mimic promoted apoptosis.

The cell migration and invasion were detected by scratch and transwell. In Y79 and SO-RB50 cell lines, compared with blank and mimic control groups, the cell migration (Figure 4A,B) and invasion (Figure 4C,D) in mimic group were down-regulated (P<0.05), suggesting that miR-486-3p inhibited cell migration and invasion.

To study the mechanism of miR-486-3p in RB cells, the Targetscan7.2 was performed to predict the target gene and the potential binding sites of miR-486-3p. As shown in Figure 5A, the 3′-UTR of ECM1 contained an area that matched to the miR-486-3p sequence. Then, Y79 and SO-RB50 were co-transfected with miR-486-3p mimic and ECM1-WT or ECM1-MUT to confirm whether miR-486-3p can bind to ECM1, and the dual-luciferase reporter was used to determine the luciferase activity. In Y79 and SO-RB50 cell lines, it was found that the luciferase activity in mimic-ECM1-WT group was reduced, while no reduction was observed in that of mimic-ECM1-MUT group (Figure 5B,C, P<0.05), ECM1 was a target gene of miR-486-3p.

In the present study, the ECM1 vectors (containing 3′-UTR) were transfected into miR-486-3p-expressing RB cells, and a control group was set up. The experimental design was divided into four groups: mimic control+NC group, mimic control+ECM1 group, mimic+NC group and mimic+ECM1 group, and Western blot was performed to confirm ECM1 expression in RB cells. We found that in Y79 and SO-RB50 cell lines, compared with mimic control+NC group, ECM1 protein level was highly expressed in mimic control+ECM1 group, and lowly expressed in mimic+NC group (Figure 6A-C, P<0.05). At the same time, ECM1 protein level in mimic+ECM1 group was lower than that in mimic control+ECM1 group but was higher than that in mimic+NC group (Figure 6A-C, P<0.05). ECM1 level was inhibited by miR-486-3p mimic in RB cells, and ECM1 vectors restored the ECM1 expression.

The cell proliferation, apoptosis, migration and invasion of transformed Y79 and SO-RB50 were determined by CCK-8, clone formation assay, flow cytometry, scratch and transwell. Compared with mimic control + NC group, cell viability (Figure 6D), clones (Figure 6G,H), migration (Figure 7A,B) and invasion (Figure 7C,D) were up-regulated in mimic control+ECM1 groups and down-regulated in mimic+NC group (P<0.05). The cell viability (Figure 6D), clones (Figure 6G,H), migration (Figure 7A,B) and invasion (Figure 7C,D) in mimic+ECM1 group were higher than those in mimic+NC group and lower than those in mimic control+ECM1 group (P<0.05). In addition, the apoptosis in mimic+NC group was up-regulated as compared with mimic control+NC group, and the apoptosis in mimic+ECM1 group was higher than that in mimic control+ECM1 group but was lower than that in mimic+NC group (Figure 6E,F, P<0.05). ECM1 could promote cell proliferation, migration and invasion, and inhibit apoptosis, as well as reverse the effect of miR-486-3p on RB cells. The miR-486-3p may play a role in RB by regulating ECM1 expression.

In the present study, we found that miR-486-3p level was down-regulated in RB tissues and cells, suggesting that miR-486-3p was involved in the development of RB. Venkatesan et al. [27] also found that the miR-486-3-p expression was down-regulated in RB samples. Then, in vitro experiments were conducted, and demonstrated that miR-486-3p overexpression inhibited the proliferation, migration and invasion, and promoted the apoptosis of RB. At the same time, the downstream target gene of miR-486-3p was analyzed and verified, and found ECM1 was the target gene of miR-486-3p and ECM1 expression was suppressed by overexpressed miR-486-3p. Finally, the role of miR-486-3p combined with target gene in RB cells was observed, and found that overexpression of ECM1 partially reversed the effect of miR-486-3p on RB cells. These results support the conclusion that miR-486-3p plays a crucial role in RB progression.

Continuous proliferation of tumor cells is one of the basic biological characteristics of tumor progression [28]. In normal conditions, the cell proliferation and apoptosis are in a dynamic equilibrium state, and abnormal cell growth or death can lead to tumor or excessive tumor cells [29]. In addition, tumor metastasis is an important cause for poor prognosis of cancer patients, and important biological characteristics of tumor metastasis are cell migration and invasion [30]. Tumor cells can enter the microcirculation through the microvessels and lymphatic vessels, and invade into the surrounding tissues, resulting in tumor metastasis [31,32]. In this study, the role of miR-486-3p on the proliferation, migration and invasion of RB cells was investigated, and the result showed that overexpression of miR-486-3p could inhibit the cell proliferation, migration and invasion of RB cells. The results revealed that miR-486-3p may play an anticancer role in RB. In other studies, miR-486-3p has been reported to have an inhibitory effect on cancer, such as glioblastoma [33], oral cancer [24] and cervical cancer [25].

In addition, the occurrence of cancer is closely related to the abnormal apoptosis regulation mechanics, which may lead to the increase in the number of tumor cells [34,35]. Meanwhile, the occurrence of apoptosis is a complex process, which is strictly regulated by many genes, including pro-apoptotic genes and apoptotic suppressor genes [36]. Bax is the most widely studied pro-apoptotic protein, which can form heterodimer with Bcl-2 (an anti-apoptotic protein), thus acting as apoptotic activator [37]. Caspase-3 belongs to the apoptotic effector gene, and the activated caspase-3 will trigger a cascade reaction, leading to irreversible apoptosis [38]. In this study, it was found that miR-486-3p promoted the apoptosis of RB cells by down-regulating the Bcl-2 level, increasing the Bax level and activating caspase-3, and thereby inhibiting the malignant progression of RB.

MiRNAs participate in a variety of physiological and pathological processes by regulating their multiple target genes [39]. It has been reported that miR-486-3p plays a critical role in proliferation and metastasis by repressing various oncogenes, including DDR1 [24] and BMP2 [40]. To further clarify the mechanism of miR-486-3p in RB, the target gene of miR-486-3p was identified. In the present paper, ECM1 was identified as the functional target of miR-486-3p in RB cells. ECM1 was as a ‘biological glue’ binding to components of the dermal–epidermal junction in the framework of normal skin [41]. ECM1 was first found in osteoblasts stromal cells, and high ECM1 levels were subsequently detected in bladder cancer [42], thyroid cancer [43] and other malignant tumors [44]. Chen et al. [45] showed that ECM1 was highly expressed in hepatocellular carcinoma specimens, and could promote the migration and invasion of HCC cells. In cervical cancer, miR-486-3p inhibited cell proliferation by targeting ECM1 [25]. We discovered that the overexpression of miR-486-3p significantly inhibited the ECM1 expression in RB cells, ECM1 could partially reverse the effects of miR-486-3p on RB cells, and promote cell proliferation, while inhibit apoptosis. The above results indicated that miR-486-3p may play a tumor suppressive role in RB through inhibiting the ECM1 expression.

In conclusion, miR-486-3p expression was decreased in RB tissues and participated in the development of RB. Moreover, miR-486-3p inhibited the cell proliferation, migration and invasion, and promoted apoptosis by inhibiting ECM1 expression, thus inhibiting the malignant progression of RB. The present study suggests that miR-486-3p may be a potential therapeutic target for clinical treatment of RB.

The authors declare that there are no competing interests associated with the manuscript.

This work was supported by the National Natural Science Foundation of China [grant number 81372878].

Substantial contributions to conception and design: H.Y. and Y.H. Data acquisition, data analysis and interpretation: J.H., G.C. and Y.D. Drafting the article or critically revising it for important intellectual content: A.W., D.G. and Y.H. Final approval of the version to be published: all authors. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of the work are appropriately investigated and resolved: H.Y.

Bax

Bcl2-associated X

Bcl-2

B-cell lymphoma-2

BMP2

bone morphogenetic protein-2

C caspase-3

cleaved caspase-3

CCK-8

cell counting kit-8

DDR1

discoidin domain receptor-1

DRAM2

DNA-damage-regulated autophagy modulator protein

ECM1

extracellular matrix protein 1

FBS

fetal bovine serum

GAPDH

glyceraldehyde-3-phosphate dehydrogenas

HRP

horseradish peroxidase

miRNA

microRNA

OD

optical density

qRT-PCR

quantitative real-time polymerase chain reaction

RB

retinoblastoma

3′-UTR

3′-untranslation region