Mechanisms Promoting Tumor Progression and Angiogenesis in Retinoblastoma: OTX2 Enhances RTN4 Transcription.

Abstract

Retinoblastoma (RB), the most prevalent intraocular cancer in children, has complex pathogenesis resulting from various genetic interactions. Research revealed that orthodenticle homeo box 2 (OTX2) has certain connection with reticulon-4 (RTN4) in regulating the angiogenesis in RB, but the molecular mechanism has not been borne out yet. This study employed an array of in vitro techniques to explore the OTX2/RTN4 interaction and its effects on RB. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and dual-luciferase reporter assays were performed to assess gene and protein expression levels. Functional impacts were evaluated through cell culture, transfection, cell viability, clone formation, wound scratch, Transwell, and in vitro tube angiogenesis assays. These methods specifically unveiled the roles of siRNA-mediated RTN4 knockdown (siRTN4), short hairpin RNA-mediated OTX2 knockdown (shOTX2), and OTX2 overexpression in modulating cellular behaviors indicative of tumorigenesis and angiogenesis. Our results demonstrated that OTX2 positively regulated RTN4, thus promoting RB cell proliferation, migration, invasion, and angiogenesis, which was significantly attenuated by knockdown of OTX2 or RTN4, but enhanced by OTX2 overexpression. OTX2 overexpression also counteracted the inhibitory effects of RTN4 knockdown on angiogenesis and tumor dynamics. In conclusion, the OTX2/RTN4 axis plays a critical role in the progression of RB by promoting malignant cellular phenotypes and angiogenesis.