Inhibiting the Progression of Human Retinoblastoma Cell by Downregulation of MMP-2/MMP-9 Using Short Hairpin RNAs (shRNAs) In Vitro

Objective To investigate the effect of downregulated matrix metalloproteinases (MMPs) gene on the proliferation, apoptosis, cell cycle, migration, and invasion of human retinoblastoma (RB) cell line in vitro. Methods Small hairpin RNA (shRNA) targeting MMP-2/MMP-9 was designed and transfected into WER1-Rb-1 cells. 48 hours after transfection, qRT-PCR and western blot technique were used to investigate the inhibitory effect of MMP-2 and MMP-9 shRNAs. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle arrest was detected using a flow cytometer while apoptosis was tested with Annexin V/PI kit. Transwell chamber assay was performed to detect the migration and invasion ability of the WER1-Rb-1 cells. Results After transfection of MMP-2/MMP-9 shRNA, there was a significant decrease in the expressions of both mRNA and protein in the shRNA groups compared with the negative and vector controls. The results of MTT assay suggested that the cell viability was significantly decreased in shRNA groups (p<0.05). Cell apoptosis also increased significantly in shRNA groups compared with the negative and vector controls (p<0.05). The flow cytometer analysis proved that the proportion of the G1 phase increased and the proportion of the G0 phase reduced significantly by the transfection of MMP-2/MMP-9 shRNA (p<0.05). The migration and invasion ability were also significantly decreased in the groups of MMP-2/MMP-9 shRNA (p<0.05). Conclusions Cell viability, migration, and invasion ability of RB cells are inhibited, and apoptosis is induced after downregulation of MMP-2/MMP-9 through RNA interference. MMP-2 and MMP-9 may be potential targets in the gene therapy of RB.


Introduction
Developing from the immature cells of retina, retinoblastoma (RB), as the most common primary intraocular malignancy of childhood, is a rare form of ocular malignant tumor [1][2][3] with an incidence of approximately 1/ 15,000-20,000 live births worldwide [4]. If diagnosed early, RB can be effectively treated by a reasonable therapy such as intravenous chemotherapy together with focal therapy (laser cryotherapy). However, most advanced cases will be involved in a distant invasion or metastasis due to the limited available diagnostic approaches, which may result in an elevated mortality rate [5]. Since the treatment of RB is mainly aimed at survival and the globe salvage and visional preservation are secondary goals, there is a critical need for the development of new targeted therapies to decrease the invasion and metastasis of RB.
Matrix metalloproteinases (MMPs) can degrade the base membrane, which is a crucial step in the tumor invasion and subsequent distant metastasis [6]. MMPs belong to a family of zinc ion-dependent endopeptidases among which MMP-2 and MMP-9 are considered to be indispensable for the degradation of the main components of extracellular matrix (ECM) [7]. Recently, the high expression of MMP-2 and MMP-9 has been confirmed in various malignant tumors, including pancreatic cancer [8], oral squamous cell carcinoma [9], cervical carcinoma [10], and RB as well [11][12][13]. e tissue inhibitors of metalloproteinases (TIMPs) are responsible for regulating the enzymatic activities of MMPs [14]. Accordingly, downregulation of the expression of the MMP-2/MMP-9 gene may be beneficial to inhibit the development and invasion of RB. However, studies on how to inhibit RB cells by silencing the MMP-2/MMP-9 gene are limited. In the present study, the expression of the MMP-2/ MMP-9 gene was downregulated by small hairpin RNA (shRNA) to study its effect on RB cell proliferation, apoptosis, cell cycle, migration, and invasion, and furthermore to explore the potential clinical significance of blocking MMP-2 and MMP-9 gene expression as an antitumor therapeutic target.

Retinoblastoma Cells.
A human RB cell line, WER1-Rb-1, was purchased from the cell bank of Shanghai Institutes for Biological Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Sigma, St. Louis, Missouri, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin, and maintained at 37°C/5% CO 2 . e medium was changed every 3 days, and cells were subcultured every 2-3 days and observed daily.

Silence of MMP-2/MMP-9 Expression in WER1-Rb-1
Cells. To suppress the MMP-2/MMP-9 expression in RB cells, a powerful gene-specific silencing technique utilizing the RNA interference (RNAi) mechanism was used. e sequence-verified plasmid vector (plenty GFP Puro) based on the short hairpin RNA library targeting human MMP-2/MMP-9 (MMP-2/MMP-9-shRNA) was purchased from GenePharma Co. (Shanghai, China). Short hairpin RNAs (shRNAs) were processed into siRNAs intracellularly, leading to a specific loss of function of MMP-2/MMP-9 gene effectively. In the experiments of transfection, WER1-Rb-1 cells were seeded into a 24-well plate and cultured in RPMI-1640 without antibiotics. And then, the transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Migration and Invasion Assay.
Transwell chamber was applied to perform the migration and invasion assay. Matrigel was used to precoat the membrane of transwell to simulate a matrix barrier for the invasion assay. e transfected cells growing in the log phase were seeded on the upper champers at a density of 1 * 10 5 cells/well. To stimulate cell invasion, 600 μl medium with 20% FBS was added to the lower chamber. After 24 h of incubation, cells which migrated to the lower chamber were fixed with paraformaldehyde for 30 min and stained with 0.1% crystal violet for 15-30 min. e images of cells were photographed with an inverted microscope (Olympus) at ×100 magnification, and the cell numbers were counted in five random fields per membrane.

Statistical
Analysis. GraphPad Prism 6 (GraphPad Software, Inc, San Diego, CA, USA) software was applied for statistical analysis. Comparisons between the groups were analyzed with two-tailed Student's t-test or one-way ANOVA. A p value <0.05 was considered statistically significant in all analyses. All data are represented as the mean ± SEM (standard error of the mean) from at least three independent experiments, as indicated with the significance score ( * < 0.05; * * < 0.01; * * * < 0.001; * * * * < 0.0001) in the figure legends.

Inhibition of MMP-2/MMP-9 Affected the Cell Cycle
Arrest and Increased Apoptosis of WER1-Rb-1 Cell. FACS was conducted to determine the effect of downregulated MMP-2/MMP-9 on the cell cycle of WER1-Rb-1 cells. As illustrated in Figures 3(a) and 3(b), the vector transfection did not influence the cell cycle, while transfection of shMMP-2/shMMP-9 after 48 hours significantly decreased the proportion of G1 phase cells compared with the vector group (vector versus shMMP-2, p � 0.0074; vector versus shMMP-9, p � 0.0105). Simultaneously, the proportion of G2 phase cells was remarkably increased 48 hours after transfection (vector versus shMMP-2, p < 0.0001; vector versus shMMP-9, p � 0.0006; Figures 3(a) and 3(c)). In addition, an FACS analysis showed that cell apoptosis rate was unaffected in the vector group in comparison to the control group, while knockdown of MMP-2/MMP-9 significantly increased the cell apoptosis rate (vector versus shMMP-2, p � 0.0034; vector versus shMMP-9, p � 0.0023; Figures 4(a) and 4(b)).

Discussion
Enucleation is the critical therapy for intraocular RB [1], which has been proved to be a therapeutic standard for the unilateral RB cases. Currently, more and more novel managements for advanced RB are being tested to minimize the need of enucleation because child survival is still the therapeutic goal with highest priority and prompt removal of affected eyeballs shows the decreasing risk of potential tumor spread [1,2,15]. e pathogenesis of tumor spread and metastasis involves a series of complicated steps, among which the degradation of the ECM is supposed to be the crucial one [16,17]. MMPs are the main proteinases, which are responsible for degrading the majority of protein in base membrane and ECM. Among the components of ECM, gelatinase A and gelatinase B which mostly degrade gelatin and several types of basal membrane collagen have a close positive correlation with MMP-2 and MMP-9, respectively [18]. Except for the degeneration of ECM, MMP-2 and MMP-9 also influence the adherence and motility of tumor cells [19]. Long H's study showed that the expression of MMP-2 and MMP-9 were closely associated with optic nerve invasion and clinical stage of RB, which implied that inhibition of MMP-2 and MMP-9 will be beneficial to the invasion and metastasis of RB [12].
Previous research has proved that MMP-2 and MMP-9 are present in RB cells, and the expression level is significantly higher in RB tissue compared to normal retina [7,[11][12][13]. In this study, we addressed the effect of downregulated MMP-2/MMP-9 gene on the proliferation, apoptosis, cell cycle, migration, and invasion of WER1-Rb-1 cell line. Besides, we can draw that the decreased cell proliferation of RB attributes to the elevated apoptosis rate from the results of cell cycle and apoptosis analysis. To the best of our knowledge, we are the first to knockdown MMP-2/ MMP-9 in RB cell in vitro stably by shRNA gene silencing technique, which was different from the study of Webb et al.

Control
Vector shMMP-2 shMMP-9 in 2017 [13]. For the past decades, RNA interference (RNAi) has been becoming a cogent tool for the silence of gene function in mammalian cells. Among the RNAi technologies, shRNAs can be applied to generate the knockdown cell lines stably, which eliminates the need for repetitive transfection and significantly improves * * * * * * * * * reproducibility of the results compared with the small interfering RNAs (siRNAs) [20]. Consistent with some previous reports [12,13], our results also indicated that cell viability, migration, and invasion ability of RB cell were inhibited, and apoptosis of RB cell was induced due to downregulation of MMP-2/MMP-9, suggesting that MMP-2   and MMP-9 might be the potential targets for preventing the progression of RB.
However, the present study still has its limitations. e molecular mechanisms by which MMP-2/MMP-9 which exert their effects on the biological behavior of RB tumor cells were not detected in this study. To get deeper understanding of MMP-2/MMP-9 effects on RB, STRING database (version 11.0) was applied to explore the potential protein-protein interactions (PPI). As demonstrated in the PPI networks of Figures 6(a) and 6(b), many proteins (described in the figure legends in detail) are closely associated with MMP-2/MMP-9. Moreover, possible coexpression, cooccurrence, metabolic pathways, and signal transduction pathways about MMP-2/MMP-2 with other proteins are obtained from the database as well.
In summary, our findings revealed that downregulation of MMP-2/MMP-9 induces the apoptosis and impairs the viability, migration, and invasion of RB tumor cells, providing a potential MMP target therapy in RB patients.

Data Availability
All data generated or analyzed during this study are included within this article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.