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Abstract

BACKGROUND AND OBJECTIVES:

MicroRNA (miR-221/222) is frequently overexpressed in many cancers and is associated with poor prognosis. However, the role of miR-221/222 in retinoblastoma (RB) remains unclear. This study aimed to detect the clinical significance of miR-221/222 in RB patients and explore its role in RB cells in vitro.

METHODS:

Expression of miR-221/222 was assessed in fresh RB tissue collected from 64 eyes and normal retinal tissues from 18 unrelated donor cadaver eyes by quantitative real time RT-PCR analysis (qRT-PCR), and correlated with the histopathological findings. Human RB Y79 cells were transfected with miR-221/222 precursors or inhibitors to overexpress or downregulate miR-221/222 expression, respectively, using Lipofectamine 2000 reagent. The biological effects of miR-221/222 were then assessed by cell viability assays, colony formation assays, apoptosis detection assays, Matrigel ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}{\textregistered}}$ invasion assays, and wound-healing assays.

RESULTS:

Higher miR-221/222 expression was detected in RB tissues compared to that of the normal retinal tissues ( $p<$ 0.001). Higher miR-221/222 expression was correlated with invasion in patients with RB. Targeting of miR-221/222 induced apoptosis and inhibited Y79 cell proliferation, migration, and invasion in vitro. However, overexpression of miR-221/222 promoted Y79 cell proliferation, migration, and invasion in vitro.

CONCLUSIONS:

Overexpression of miR-221/222 was associated with tumor invasiveness in patients with RB. The miR-221/222 cluster might be used as a potential therapeutic strategy in clinical practice.

Keywords

Retinoblastoma miR-221/222 marker invasion

1. Introduction

Retinoblastoma (RB) is a childhood cancer that arises from the primitive retinal layer. Despite a good understanding of its etiology, mortality from RB is approximately 70% in countries of low and middle income. Worldwide, most of the estimated 9,000 newly diagnosed patients every year will unfortunately die [1]. The current management is enucleation (removal of the eye in childhood), chemotherapy, and/or focal therapy [2]. However, each of these therapies has major drawbacks for pediatric patients. Therefore, targeted therapy is gaining importance in the management of RB [3].

In recent years, microRNAs (miRNAs) have received increasing attention in cancer research. These small, non-coding RNAs can inhibit target gene expression by binding to the 3’-untranslated region (3’-UTR) of target mRNA, resulting in either mRNA degradation or inhibition of translation to protein. MiRNAs play essential roles in many normal biological processes involving cell proliferation, differentiation, apoptosis, and stress resistance [4, 5]. Studies have also shown that aberrant miRNA expression is correlated with the development and progression of cancers, thus, miRNAs could be used as biomarkers for the diagnosis and prognosis of cancers. However, the miRNAs can have oncogenic or tumor suppressor activities, and therefore, miRNAs are emerging as targets for cancer molecular therapy [6].

MiR-221 and miR-222 (miR-221/222) are expressed from a single transcript that is found on the X chromosome. Both miRNAs possess the same sequence, which is an evolutionarily conserved, short region at the 5’ end through which they bind target sites in the mRNA’s 3’ UTR. As oncogenes, miR-221/222 has been studied in epithelial cancers, including glioma, prostate carcinoma, hepatocellular cancer, lung cancer, and breast cancer [7, 8, 9, 10, 11]. The expression of miR-221 is significantly upregulated in pancreatic cancer cell lines and tumor tissues compared with normal pancreatic duct epithelial cells and normal pancreatic tissues [10]. Furthermore, overexpression of miR-221/222 may play an oncogenic role in pancreatic cancer by inducing the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, thus leading to cancer cell invasion [11]. Therefore, high levels of miR-221/222 may be useful as prognostic markers, and thus could aid in the development of some new diagnostic and prognostic methods for cancer treatments. The inhibition or silencing of miR-221/222 may also provide novel treatment methods for cancers. However, the relationship between the miR-221/222 expression and clinicopathological parameters in patients with RB is not fully understood.

In the present study, we investigated the expression of miRNA-221/222 in RB tissues and analyzed the correlation between miR-221/222 expression and clinicopathological parameters of RB. We also performed in vitro experiments to study the effects of miR-221/222 on cell growth, apoptosis, and invasion in cultured RB cell lines.

2. Materials and methods

2.1 RB tumor and normal retinal specimens

This study was approved by the Affiliated Hospital of Qingdao University, Qingdao, China. As the patients were all under the age of 5 years, the parent(s) or legal guardians signed the consent forms. Diagnosis of RB was established by standard ophthalmic and histological criteria. Fresh RB tissue was collected from the 64 eyes (48 unilateral RBs and 16 bilateral RBs) according to the ethical and legal standards of our institute. In brief, the tumor tissue was harvested following enucleation. The globe was opened with an 8.0-mm corneal trephine so that the trephine straddled a margin of the tumor within the eye. The sclera was carefully opened so that seeding was avoided, and the choroidal tissue was incised. The RB tumor was visualized, and tissue was obtained using a 6-mm tissue spoon and Westcott scissors. We obtained normal retinal tissues from 18 unrelated donor cadaver eyes as controls. All specimens were flash frozen on dry ice and transferred for miR-221/222 analyses. The globe and scleral cap were placed in formalin and sent to the pathology laboratory. The protocol for miR-221/222 analysis of RB tissues was approved by the Institutional Review Board of the Affiliated Hospital of Qingdao University.

2.2 Cell culture

Human RB Y79 cells were purchased from the American Type Culture Collection (ATCC, Shanghai, China). Cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI; Gibco-BRL, Rockville, MD, USA) supplemented with 10% heat-inactivated fetal calf serum (FBS; Gibco-BRL), 0.1% ciprofloxacin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 4.5% dextrose (Sigma-Aldrich, St. Louis, MO, USA) and grown in suspension at 37 ${}^{\circ}$ C in 5% CO ${}_{2}$ .

2.3 In vitro transient or stable transfection of Y79 cells by miR-221/222 precursors or inhibitors

To manipulate the cellular functions of Y79 cells, miR-221/222 was transiently transfected into Y79 cells with 100 nM miR-221/222 precursors or miR-221/222 inhibitors for 48 h to increase or decrease miR-221/222 expression, respectively, using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Non-targeting miR-221/222 precursors (precursor controls) or miR-221/222 inhibitors (inhibitor controls) were used as the controls. The transfection efficiency was confirmed by quantitative real time RT-PCR (qRT-PCR).

To produce stable transfectants, the transiently transfected Y79 cells were selected by culturing in puromycin (4 $\mu$ g/mL) for 2–3 weeks to generate miR-221/222 precursors or miR-221/222 inhibitors, or negative control expressing clones. Successful transfection was assessed by measuring miR-221/222 expression using qRT-PCR. Single clones were isolated and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS and 1% penicillin-streptomycin (Invitrogen) at 37 ${}^{\circ}$ C in a 5% CO ${}_{2}$ incubator.

2.4 Quantitative real time RT-PCR analyses

Total RNA isolation from cells transfected as previously described was carried out with TriPure isolation reagent (Roche, Indianapolis, IN, USA). The cDNA synthesis was performed by incubation with 100 ng RNA, 1 $\times$ RT buffer, 0.25 mM of each dNTPs, 0.25 U/ $\mu$ L RNase-OUT inhibitor, and 3.33 U/ $\mu$ L M-MLV reverse transcriptase, in a one step 7.5 $\mu$ L volume. The reaction was incubated in a 384-well plate at 37 ${}^{\circ}$ C during 30 min using an Applied Biosystems 7900 Real-Time PCR Instrument (Applied Biosystems, Foster, CA). For analysis of miR-221/222 expression, real time RT-qPCR analyses were performed using TaqMan pri-miRNA assays (Applied Biosystems) according to the instruction manual. Ct values were analyzed to determine the statistical significance of miR-221/222 expression in Y79 cells. Relative expression was calculated using the 2 ${}^{-\Delta\Delta\text{Ct}}$ method and normalized to the expression of RNU6 (Applied Biosystems). The reaction was incubated in a 384-well plate in an Applied Biosystems 7900 Real-Time PCR Instrument. All RT-qPCRs were performed in triplicate.

2.5 Flow cytometry analysis

Y79 cells were transiently transfected with 100 nM miR-221/222 precursors or miR-221/222 inhibitors or negative controls for 48 h. Then, the cells were collected and pelleted at 1,500 rpm for 3 min and suspended in 1 mL of hypotonic fluorochrome solution containing 50 $\mu$ g/mL propidium iodide (PI) and annexin in 0.1% sodium citrate plus 0.1% Triton X-100, then analyzed by flow cytometry (ESP Elite, Coulter, Miami, FL, USA).

2.6 Cell viability assay

Cell viability was analyzed using the 3-(4, 5-dim- ethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). Briefly, Y79 cells (5 $\times$ 10 ${}^{3}$ ) were plated onto 96-well plates (NEST ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}{\textregistered}}$ ) in 100 $\mu$ L of growth medium and allowed to adhere overnight. The cells were then transfected with 100 nm of miR-221/222 precursors or miR-221/222 inhibitors or negative controls. After 48 h, the culture medium was removed and replaced with culture medium containing 10 $\mu$ L of sterile MTT reagent (5 mg/mL). After incubation at 37 ${}^{\circ}$ C for 4 h, the MTT solution was removed, and 150 $\mu$ L of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. Spectrometric absorbance at 490 nm was measured by a BioTek ELx800 ${}^{\text{TM}}$ microplate photometer (BioTek EL $\times$ 800, SN211805; Thermo Fisher, Scotts Valley, CA, USA).

2.7 Clonogenic assay

Soft agar assays were performed according to the manufacturer’s protocol. An equal volume of 2 $\times$ medium containing 20% FBS and 1% agar were mixed to obtain the underlay. The top agar layer was prepared using medium containing 0.35% agar. The cells (1 $\times$ 10 ${}^{5}$ cells; stable transfectants) were suspended in the top agar. Cells were seeded in the 60 mm culture dishes containing the underlay. Two mL of complete medium was supplemented twice a week. Cells were cultured for 3 weeks, after which colonies were quantified in triplicate (number $\pm$ SEM of cells per field, 10 $\times$ magnification).

2.8 Matrigel ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}{\textregistered}}$ invasion assay

For migration assays, the stable miR transfected Y79 cells were resuspended in 200 $\mu$ L of serum-free DMEM and seeded onto Boyden chambers (Corning, Corning, NY, USA) with an 8.0- $\mu$ m pore membrane (5 $\times$ 10 ${}^{4}$ cells/well). The chambers were then incubated in DMEM with 20% FBS at 37 ${}^{\circ}$ C in 5% CO ${}_{2}$ . After 24 h, the cells adhering to the chamber’s lower surface were fixed, whereas cells remaining on the upper surface were removed. After staining in 0.05% Crystal Violet, the cells from three randomly selected high power fields were counted under a microscope (Olympus, Tokyo, Japan). Invasion ability was evaluated by counting the migrated cells in five randomly selected fields, under a light microscope. Migration assays were conducted in triplicate.

2.9 Wound-healing assay

A CytoSelect ${}^{\text{TM}}$ 24-well Would Healing Assay kit was purchased from Cell Biolabs Inc. (Shanghai, China). The 24-well plate was pretreated with 500 $\mu$ L of 0.1 mg/mL poly-L-lysine hydrobromide (Sigma-Aldrich) as per the manufacturer’s instructions, and incubated at 37 ${}^{\circ}$ C for 1 h. The wells were washed with distilled sterile water twice and dried in a biosafety cabinet for 2 h. Then, the stably miR-transfected Y79 cells (miR-221/222 precursors, miR-221/222 inhibitors, or negative controls) were plated in the wells at a concentration of 5 $\times$ 10 ${}^{4}$ cells/mL per well. We ensured that the cells were evenly distributed, and incubated the plate at 37 ${}^{\circ}$ C to create a 60%–70% confluent monolayer of cells. The inserts were removed, and the wells were washed twice with distilled sterile water to remove unattached cells and debris. A scratch was made using a 10- $\mu$ L sterile pipette tip to create a 900 $\mu$ m wound. The wounded monolayer was washed with phosphate-buffered saline (PBS) and imaged at 0 h and 24 h after scratching, using a microscope (Leica, Wetzlar, Germany) equipped with a CCD camera (Leica). The migration percentages were calculated according to the equation: percentage wound-healing $=$ [(wound length at 0 h) $-$ (wound length at 24 h)]/(wound length at 0 h) $\times$ 100. The mean results of three straight distances (upper edge, middle, and lower edge) in the scratch area were evaluated as wound lengths. Length quantification was performed using ImageJ software (NIH, Bethesda, MD, USA). Wound-healing assays were conducted in triplicate.

2.10 Statistical analysis

Statistical significance of the data was assessed using SPSS, version 13.0 for Windows (SPSS Inc., Chicago, IL, USA). The median value for miR-221/222 was used as a cutoff value, and the expression level of miR-221/222 was categorized as low or high. Nonparametric Kruskal-Wallis tests and Mann-Whitney U tests were conducted to compare the expression levels of miR-221/222 in RB and control samples. The chi-square test or Fisher’s exact test was used for analyzing miR-221/222 expression (low vs. high) with clinicopathological characteristics of tumors. Differences in the median values among different groups were analyzed by one-way analysis of variance (ANOVA) and a post hoc Dunnett’s test. A value of $p<$ 0.05 was regarded as statistically significant.

3. Results

3.1 Quantification of miR-221/222 levels in RB tissues

We first examined miR-221/222 expression in 64 RB tissues and 18 normal retinal tissues. The mean age (months) $\pm$ (SD) was 21.43 $\pm$ 10.62 in patients with RB. There were significant differences in the expression of miR-221/222 in the RB tissues and normal retinal tissues. The miR-221/222 expression level was significantly higher in the RB tissues than that of the normal retinal tissues ( $p<$ 0.001) (Fig. 1).

Figure 1.

Difference in microRNA-221/222 (miR-221/222) expression levels in retinoblastoma (RB) and normal tissues. The relative expression level of miR-221/222 was normalized to RNU6. The bold line represents the median value. Statistically significant differences were determined using the Mann-Whitney U tests. The results revealed a higher level of miR-221/222 in RB tissues ( $p<$ 0.001) (NC: normal retinal tissues).

3.2 MiR-221/222 expression and clinicopathological features in RB

We next evaluated the relationship between the expression of miR-221/222 and clinicopathological features of RB (Table 1). The relative median miR-221 and miR-222 values were 0.0528 and 0.0794 in normal retinal tissues, respectively (Fig. 1). Therefore, we used 0.0528 and 0.0794 as the cutoff values. The RB patients were then divided into group A ( $n=$ 41 and $n=$ 36) as those with the higher miR-221 and miR-222 levels ( $\geqslant$ 0.0528 and $\geqslant$ 0.079) and group B ( $n=$ 23 and $n=$ 28) as those with lower miR-221 and miR-222 levels ( $<$ 0.0528 and $<$ 0.079). The $\chi$ 2 analyses showed that higher miR-221 and miR-222 levels correlated well with extents of invasion ( $p=$ 0.015 and $p=$ 0.018, respectively). There was no significant difference between these groups in terms of pattern of tumor growth and degree of differentiation.

Table 1
MiR-221/222 expression and clinicopathological characteristics in subjects with retinoblastoma ( $n=$ 64)

Variable	High	Low	$p$ -value	High	Low	$p$ -value
	miR-221 expression			miR-222 expression
Sex			0.517			0.179
Male ( $n=$ 29)	19	10		14	15
Female ( $n=$ 35)	22	13		22	13
Tumor laterality			0.446			0.616
Unilateral ( $n=$ 48)	30	18		27	21
Bilateral ( $n=$ 16)	11	5		9	7
Pattern of tumor growth			0.203			0.318
Endophytic ( $n=$ 23)	14	9		12	11
Exophytic ( $n=$ 6)	3	3		2	4
Mixed ( $n=$ 35)	24	11		22	13
Degree of differentiation			0.113			0.225
Well/Moderate ( $n=$ 41)	29	12		25	16
Poor ( $n=$ 23)	12	11		11	12
Choroidal invasion			0.014			0.017
Negative ( $n=$ 26)	12	14		10	16
Positive ( $n=$ 38)	29	9		26	12
Optic nerve invasion			0.004			0.003
Negative ( $n=$ 35)	17	18		15	20
Positive ( $n=$ 29)	24	5		21	8
Extent of Invasion			0.015			0.018
No invasion beyond retina ( $n=$ 16)	5	11		6	10
Choroidal invasion only ( $n=$ 15)	9	4		6	9
Optic nerve invasion only ( $n=$ 8)	4	4		5	3
Both choroidal and optic nerve invasion ( $n=$ 25)	21	4		19	6

3.3 MiR-221/222 affected cell viability in Y79 cells in vitro

To determine the function of miR-221/222, we applied miR-221/222 precursors and inhibitors to increase or decrease miR-221/222 expression, respectively, in Y79 cells. Upon transient transfection with miR-221/222 precursors for 48 h, the expression level of miR-221/222 increased more than 3.6-fold/2.8-fold compared with that of the untreated cells. In contrast, the expression level of miR-221/222 decreased up to 60%/70% when miR-221/222 inhibitors were transiently transfected in the Y79 cells for 48 h (Fig. 2A).

To investigate the influence of miR-221/222 on cell viability, the MTT assay was used. MTT assay results showed that cell viability was significantly increased in Y79 cells after transient transfection with miR-221/222 precursors for 48 h, and was significantly decreased in Y79 cells after transfection with miR-221/222 inhibitors for 48 h (Fig. 2B).

We next tested whether miR-221/222 precursors or miR-221/222 inhibitors affected the colony forming ability of Y79 cells. MiR-221/222 precursors or miR-221/222 inhibitors were stably transfected into the Y79 cells. The miR-221/222 expression was detected in the stable miR-221/222 precursors or miR-221/222 inhibitors transfected Y79 cells by qRT-PCR. The results showed that miR-221/222 expression was significantly increased in the stable miR-221/222 precursors transfected Y79 cells (Fig. 2C), and significantly decreased in the stable miR-221/222 inhibitors transfected Y79 cells (Fig. 2C), indicating that the miRs were successfully transfected in the Y79 cells.

Soft agar assay results showed that Y79 cells in the untreated groups formed large numbers of colonies, whereas cells transfected with miR-221/222 inhibitors formed very few colonies (Fig. 2D and E). The cells transfected with miR-221/222 precursors formed very large colonies compared to that of the untreated cells (Fig. 2D and E).

Figure 2.

Effect of miRs on Y79 cell proliferation. A, The miR-221/222 precursor or inhibitor transfection efficiency in transiently transfected Y79 cells was confirmed by qRT-PCR. B, Cell viability was assessed by MTT assays after transfection with miR-221/222 precursors or miR-221/222 inhibitors. C, The miR-221/222 precursor or inhibitor transfection efficiency in stably transfected Y79 cells was confirmed by qRT-PCR. D, Colonies after transfection with miR-221/222 precursors or miR-221/222 inhibitors were stained with Crystal Violet, followed by counting and scoring of colony numbers. E, Representative images of Y79 cell colonies stained with Crystal Violet. F, Cells were stained with annexin V-FITC/PI, and apoptosis was analyzed by flow cytometry. G, The percentage of apoptotic cells is reported. Data are presented as the mean $\pm$ SD of two independent experiments ( ${}^{*}p<$ 0.01). PI $=$ propidium iodide.

Figure 3.

Effect of miR-221/222 on cell invasion and migration in vitro. A, Transwell ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}{\textregistered}}$ invasion assay results from different treatment groups with representative images of the invasion assays for Y79 cells. B, Comparisons of the invasive cell counts in different groups of Y79 cells. C, Scratch wound-healing assay results of different treatment groups. Representative images of the wound-healing assay results for Y79 cells. D, Comparisons of migration distances in Y79 cells. ${}^{*}p<$ 0.05, ${}^{**}p<$ 0.01, ${}^{\#}p<$ 0.05. Data are shown as the means $\pm$ SD from three independent experiments.

3.4 Targeting miR-221/222 induced apoptosis in Y79 cells

To investigate the effects of miR-221/222 precursors or miR-221/222 inhibitors on apoptosis, PI and annexin V double staining assays were performed (Fig. 2F and G). At 48 h post-transfection, a significantly higher apoptotic levels was observed in the miR-221/222 inhibitors transfected Y79 cells compared with that of the untreated controls, suggesting that miR-221/222 inhibitors promoted cell apoptosis. In addition, less cell apoptosis was shown in the untreated Y79 cells, and miR-221/222 precursor transfection did not significantly affect cell apoptosis in Y79 cells (data not shown).

3.5 MiR-221/222 modulated cell invasion and migration in Y79 cells in vitro

To investigate whether miR-221/222 was associated with cell invasion, we transfected Y79 cells with miR-221/222 precursors or inhibitors. Based on the Matrigel ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}{\textregistered}}$ invasion assay results (Fig. 3A), the invasion ability of miR-221/222 precursor transfected cells was significantly increased compared with the negative control cells. Moreover, miR-221/222 inhibitor transfection significantly decreased cell invasion compared with that of the cells treated with negative control (Fig. 3A and B). A wound healing assay performed using Y79 cells revealed that the miR-221/222 precursors and miR-221/222 inhibitors had the same trends as the Matrigel ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}{\textregistered}}$ invasion assay results (Fig. 3C and D). Together, the results showed that miR-221/222 overexpression promoted cell invasion, and targeting miR-221/222 inhibited cell invasion.

4. Discussion

MiR-221 and miR-222, encoded in tandem from a gene cluster located on the X chromosome (Xp11.3), contain identical seed sequences separated by 727 bases, and are highly conserved in vertebrates [12]. In healthy conditions, they have been found to regulate essential physiological vascular processes such as angiogenesis, neointimal hyperplasia, vessel wound-healing, vascular aging, and atherosclerotic vascular remodeling [13, 14, 15, 16]. These two highly homologous miRNAs frequently act as a gene cluster (miR-221/222). Recently, it has found that miR-221/222 was overexpressed in breast cancer [17], gastric cancer [18], colorectal cancer [19], and pancreatic cancer [20]. Furthermore, miR-221/222 is almost undetectable in normal human melanocytes and is found to be increasingly expressed in cancers [21]. MiR-221/222 may therefore mediate the functions of cancer cells to proliferate, differentiate, and invade [17, 18, 19, 20].

Our results are the first to demonstrate that miR-221/222 expression was significantly higher in the RB tissues than that in the normal retinal tissues, as determined by qRT-PCR. Moreover, high levels of miR-221/222 in RB tissues were associated with choroidal invasion, optic nerve invasion, and extent of invasion. These data suggested that miR-221/222 overexpression is common in human RB and may have a strong association with carcinogenesis and invasion.

Considering the important role of miR-221/222 in RB, we observed the cell behavior of Y79 cells in vitro. The MTT and clonogenic assay results showed that cell viability and cell survival were significantly inhibited by silencing endogenous miR-221/222, but the recovery of the miR-221/222 levels could relieve these phenomena and further stimulate tumor cell survival. To further explore the potential mechanisms underlying the decreased cell viability and growth of Y79 cells, cell apoptosis was analyzed by flow cytometry. The miR-221/222 inhibitor transfected cells showed an increase in the number of apoptotic cells compared with that of the negative control transfected cells. However, several studies reported tumor suppressive functions of miR-221/222. One study found that overexpression of miR-221/222 in malignant glioblastoma cells increased the population of cells in S-phase, resulting in massive apoptosis [22]. In addition, another study reported that miR-221 and miR-222 were downregulated in Kaposi sarcoma-associated herpes virus-associated cancers, including primary effusion lymphoma and Kaposi sarcoma [23]. The results of our study showed that miR-221/222 regulated cell growth and apoptosis depending on the tissue specificity.

Previous studies have reported that overexpression of miR-221/222 promoted invasion in pancreatic cancer cells, and silencing the endogenous miR-221/222 reversed the effect [24]. In our study, we found that overexpression of miR-221/222 promoted Y79 cell migration and invasion using Transwell ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}{\textregistered}}$ assays and wound-healing assays, and restoration of miR-221 and miR-222 significantly inhibited cancer cell migration and invasion. These results strongly suggested that miR-221 and miR-222 positively influenced the invasiveness of RB cells.

In summary, we confirmed the expression of miR-221/222 in human RB tissues and their oncogenic roles in RB cells. Our data showed that miR-221/222 expression was upregulated in human RB, and high levels of miR-221/222 expression in RB tissues were significantly associated with invasion in patients with RB. We also provided direct evidence that overexpression of miR-221/222 promoted proliferation and invasion in RB cells. Silencing miR-221/222 promoted apoptosis and inhibited proliferation and invasion in RB cells, providing a robust framework for clinical development of synthetic miR-221/222 inhibitors as novel therapeutics for RB treatment.

Footnotes

Acknowledgments

This study was supported by grants from National Natural Science Foundation of China (No: 81170825).

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Upregulated miR-221/222 promotes cell proliferation and invasion and is associated with invasive features in retinoblastoma

Abstract

BACKGROUND AND OBJECTIVES:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 RB tumor and normal retinal specimens

2.2 Cell culture

2.3 In vitro transient or stable transfection of Y79 cells by miR-221/222 precursors or inhibitors

2.4 Quantitative real time RT-PCR analyses

2.5 Flow cytometry analysis

2.6 Cell viability assay

2.7 Clonogenic assay

2.8 Matrigel ® invasion assay

2.9 Wound-healing assay

2.10 Statistical analysis

3. Results

3.1 Quantification of miR-221/222 levels in RB tissues

Table 1 MiR-221/222 expression and clinicopathological characteristics in subjects with retinoblastoma ( n = 64)

3.5 MiR-221/222 modulated cell invasion and migration in Y79 cells in vitro

4. Discussion

Footnotes

Acknowledgments

References

2.8 Matrigel ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}{\textregistered}}$ invasion assay

Table 1
MiR-221/222 expression and clinicopathological characteristics in subjects with retinoblastoma ( $n=$ 64)