Abstract
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.
Highlights
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]
Matrix metalloproteinases (MMPs)-2/MMP-9 Downregulated by RNA Interference in WER1-Rb-1 Cells. e Small hairpin RNA (shRNA) sequences for MMP-2/ MMP-9 were fused with a green fluorescent protein (GFP) cDNA by using the plasmids in this study. erefore, the transfected WER1-Rb-1 cells exhibited strong green fluorescence under a fluorescence microscope, while there was no fluorescence for the control group (Figure 1(a))
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 extracellular matrix (ECM) is supposed to be the crucial one [16, 17]
Summary
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]. 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]. The high expression of MMP-2 and MMP-9 has been confirmed in various malignant tumors, including pancreatic cancer [8], oral squamous cell
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