Purpose: Circular RNAs (circRNAs) play essential roles in the progression of human tumors, including retinoblastoma (RB). In this study, we aimed to explore the functions and potential mechanisms of circ_0000527 in RB.
Methods: Quantitative real-time polymerase chain reaction (qRT-PCR), Western blot assay and immunohistochemistry (IHC) assay were conducted to determine the levels of circ_0000527, microRNA-1236-3p (miR-1236-3p) and SMAD family member 2 (SMAD2). RNase R assay and actinomycin D assay were conducted to analyze the characteristic of circ_0000527. Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, and colony formation assay were performed for cell proliferation ability. Wound healing assay and transwell assay were applied to assess cell migration and invasion. Tube formation assay was utilized for angiogenesis ability. Flow cytometry analysis was adopted to analyze cell apoptosis. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to analyze the relationships among circ_0000527, miR-1236-3p, and SMAD2. Murine xenograft model assay was conducted for the role of circ_0000527 in vivo.
Results: Circ_0000527 was overexpressed in RB patients and related to advanced TNM stages, optic nerve invasion and choroidal invasion. Circ_0000527 knockdown suppressed cell proliferation, migration, invasion and angiogenesis and promoted apoptosis in RB cells in vitro. Circ_0000527 sponged miR-1236-3p, which directly targeted SMAD2. MiR-1236-3p level was decreased in RB tissues and cells. MiR-1236-3p inhibition reversed circ_0000527 knockdown-mediated effects on RB cell malignant behaviors. Moreover, miR-1236-3p overexpression suppressed RB cell progression, with SMAD2 elevation abrogated the effect. Additionally, circ_0000527 knockdown restrained tumor formation in vivo.
Conclusions: Circ_0000527/miR-1236-3p/SMAD2 axis played a positive role in the progression of RB.
Keywords: MiR-1236-3p; Retinoblastoma; SMAD2; circ_0000527.