MiR-486-3p Inhibits the Proliferation, Migration and Invasion of Retinoblastoma Cells by Targeting ECM1

It has reported that miR-486-3p expression is decreased in retinoblastoma (RB) tumor tissues, however, its function in RB has been less reported. This study aimed to explore the regulatory effects of miR-486-3p on RB cells. The expression of miR-486-3p in RB tissues and cells was detected by quantitative real-time (qRT)-PCR. Cell viability, proliferation, apoptosis, migration and invasion ability were determined by cell counting kit-8 kit, clone formation assay, flow cytometry, scratch assay and transwell, respectively. Targetscan 7.2 and dual-luciferase reporter were used to verify target genes for miR-486-3p. The expressions of apoptosis-related proteins and ECM1 were detected by Western blot. The miR-486-3p expression was decreased in RB tissues and cells. In RB cells, overexpression of miR-486-3p inhibited cell proliferation, migration and invasion, while promoted apoptosis. Moreover, overexpression of miR-486-3p decreased Bcl-2 expression, while increased the expressions of Bax and C caspase-3. ECM1 was the target gene of miR-486-3p, and miR-486-3p inhibited the expression of ECM1. Furthermore, ECM1 partially reversed the inhibitory effect of miR-486-3p on the proliferation, migration and invasion of RB cells. MiR-486-3p inhibited the proliferation, migration and invasion of RB by down-regulating ECM1.


Introduction
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)(6)(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)(13)(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 was act as novel potential biomarkers for the detection of retinoblastoma (17). In addition, studies have found that many miRNAs are abnormally expressed in RB, such as miR-192, Downloaded from https://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20200392/879121/bsr-2020-0392.pdf by guest on 14 May 2020 Bioscience Reports. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BSR20200392 miR-34a and miR-376a (18)(19)(20)(21). miR-125b promoted tumor growth and suppresses apoptosis by targeting DRAM2 in retinoblastoma (22). miRNA-188-5p promoted epithelial-mesenchymal transition via targeting DNA binding 4 through Wnt/β-catenin Signaling in retinoblastoma (23). These studies indicate that abnormal miRNA abnormally expression 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)(25)(26). The down-regulation of miR-486-3p is considered as one of the causes for metastasis of cervical cancer (25). Venkatesan et al. found that miR-486-3p expression was decreased in the RB tumor tissues (27), 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.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from cells by Trizol reagent. RNA purity and cycles, and 72℃ for 5 min. The expression level was calculated using 2 -ΔΔCT method.
U6 was used as internal reference gene, and the primers for qRT-PCR used in this study were shown in Table 2.

Western blot
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

Statistical analysis
Experiments were repeated at least 3 times. SPSS 20.0 system (SPSS Inc., USA) was used for statistical analysis. All experimental data was presented as the mean ±standard deviation (SD). Comparisons between groups were analyzed with student's tests or one-way ANOVA. P <0.05 was considered to be statistically significant.

miR-486-3p was down-regulated in RB tissues and cell lines
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, Fig   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 (Fig 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.

miR-486-3p mimic inhibited cell proliferation
Cell viability and the ability of cell cloning were detected by CCK-8 and clone formation assay. As shown in Fig 2BC, 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 (Fig 2E, P<0.05). The cell viability and the ability of cell cloning were inhibited by miR-486-3p.

miR-486-3p mimic promoted apoptosis
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 (Fig 2F, 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 (Fig 3B, P<0.05). In addition, in SO-RB50 cell line, compared with blank and mimic control groups, the apoptosis rate ( Fig 2F) and the Bax and C caspase-3 expressions (Fig 3D) in mimic group were up-regulated, and the Bcl-2 expression (Fig 3D) 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 line, compared with blank and mimic control groups, the cell migration ( Fig 4B) and invasion (Fig 4C) in mimic group were down-regulated (P<0.05), suggesting that miR-486-3p inhibited cell migration and invasion.

ECM1 was a target gene of miR-486-3p in RB cells
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 Fig 5A,  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 (Fig 5BC, P<0.05), ECM1 was a target gene of miR-486-3p.

miR-486-3p inhibited ECM1 expression
In this 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 4 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 (Fig 6C,   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 (Fig 6C, P<0.05). ECM1 level was inhibited by miR-486-3p mimic in RB cells, and ECM1 vectors restored the ECM1 expression.

ECM1 reversed the effect of miR-486-3p on RB cells
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 (Fig 6D), clones (Fig 6H), migration ( Fig 7B) and invasion (Fig 7C) were up-regulated in mimic control + ECM1 groups and down-regulated in mimic+ NC group (P<0.05).
The cell viability (Fig 6D), clones (Fig 6H), migration ( Fig 7B) and invasion (Fig 7C) 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 to 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 (Fig 6F, 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.

Discussion
In this study, we found that miR-486-3p level was down-regulated in RB tissues 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 of 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 hetero dimer 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 this 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, Downloaded from https://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20200392/879121/bsr-2020-0392.pdf by guest on 14 May 2020 and high ECM1 levels were subsequently detected in bladder cancer (42), thyroid cancer (43) and other malignant tumors (44). Hao 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. This study suggests that miR-486-3p may be a potential therapeutic target for clinical treatment of RB.

Conflicts of Interest
The authors declare no conflicts of interest.   Y79 was co-transfected with miR-486-3p mimic and ECM1 or their negative control (mimic control+NC group, mimic control+ ECM1 group, mimic + NC group, mimic + ECM1 group), the ECM1 protein level was detected by western blot. B: SO-RB50 was co-transfected with miR-486-3p mimic and ECM1 or their negative control (mimic control+NC group, mimic control+ ECM1 group, mimic + NC group, mimic + ECM1 group), the ECM1 protein level was detected by western blot. C: The miR-486-3p mimic decreased ECM1 level, while ECM1 reversed the effect of the Downloaded from https://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20200392/879121/bsr-2020-0392.pdf by guest on 14 May 2020 Bioscience Reports. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BSR20200392