WTAP‐mediated m6A modification of circ_0032463 promotes osteosarcoma progression by sponging miR‐145‐5p and regulating GFRA1 expression

Abstract

AbstractOsteosarcoma (OS) is the most frequent bone malignancy in humans. Previous evidence suggest that circ_0032463 is an oncogenic circular RNA (circRNA) in various cancers, including OS. However, the molecular mechanism of circ_0032463 involved in OS is still unclear. Circ_0032463, microRNA‐145‐5p (miR‐145‐5p), GDNF receptor alpha 1 (GFRA1), and Wilms tumor 1‐associated protein (WTAP) levels were determined using real‐time quantitative polymerase chain reaction (RT‐qPCR). Cell proliferation, apoptosis, migration, invasion, and angiogenesis were analyzed using 5‐ethynyl‐2′‐deoxyuridine (EdU), flow cytometry, transwell, and tube formation assays. Western blot analysis was performed to measure matrix metalloproteinase 2 (MMP2), MMP9, GFRA1, and WTAP protein levels. Binding between miR‐145‐5p and circ_0032463 or GFRA1 was confirmed using a dual‐luciferase reporter and pull‐down assay. The biological role of circ_0032463 on OS cell growth was also analyzed using a xenograft tumor model in vivo. Methylated RNA immunoprecipitation assay validated the interaction between WTAP and circ_0032463. Circ_0032463, GFRA1, and WTAP levels were increased, and miR‐145‐5p was decreased in OS tissues and cells. Circ_0032463 deficiency might hinder OS cell proliferation, migration, invasion, angiogenesis, and promote apoptosis in vitro. Mechanically, circ_0032463 worked as a miR‐145‐5p sponge to increase GFRA1 expression. Repression of circ_0032463 knockdown on tumor cell growth was proved in vivo. Besides, N6‐methyladenosine (m6A) modification facilitates the biogenesis of circ_0032463. Taken together, m6A‐mediated biogenesis of circ_0032463 facilitates OS cell malignant biological behavior partly via regulating the miR‐145‐5p/GFRA1 axis, suggesting a promising molecular marker for OS treatment.