CircLRFN5 inhibits the progression of glioblastoma via PRRX2/GCH1 mediated ferroptosis

Background: Ferroptosis is a novel form of iron-dependent cell death and participates in the malignant progression of glioblastoma (GBM). Although circular RNAs (circRNAs) are found to play key roles in Ferroptosis via several mechanisms, including regulating iron metabolism, glutathione metabolism, lipid peroxidation and mitochondrial-related proteins, there are many novel circRNAs regulating Ferroptosis need to be found, and they may become a new molecular treatment target in GBM.

Methods: The expression levels of circLRFN5, PRRX2 and GCH1 were detected by qPCR, western blotting, and immunohistochemistry. Lentiviral-based infections were used to overexpress or knockdown these molecules in glioma stem cells (GSCs). The biological functions of these molecules on GSCs were detected by MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium), the 5-ethynyl-20-deoxyuridine (EdU) incorporation assay, transwell, neurosphere formation assays, Extreme Limiting Dilution Analysis (ELDA) and xenograft experiments. The content of Ferroptosis levels in GSCs was detected by BODIPY 581/591 C11 assay, glutathione (GSH) assay and malondialdehyde (MDA) assay. The regulating mechanisms among these molecules were studied by RNA immunoprecipitation assay, RNA pull-down assay, ubiquitination assay, dual-luciferase reporter assay and chromatin immunoprecipitation assay.

Results: We found a novel circRNA circLRFN5 is downregulated in GBM and associated with GBM patients' poor prognosis. CircLRFN5 overexpression inhibits the cell viabilities, proliferation, neurospheres formation, stemness and tumorigenesis of GSCs via inducing Ferroptosis. Mechanistically, circLRFN5 binds to PRRX2 protein and promotes its degradation via a ubiquitin-mediated proteasomal pathway. PRRX2 can transcriptionally upregulate GCH1 expression in GSCs, which is a Ferroptosis suppressor via generating the antioxidant tetrahydrobiopterin (BH4).

Conclusions: Our study found circLRFN5 as a tumor-suppressive circRNA and identified its role in the progression of Ferroptosis and GBM. CircLRFN5 can be used as a potential GBM biomarker and become a target for molecular therapies or ferroptosis-dependent therapy in GBM.

CircLRFN5; Ferroptosis; GCH1; GSCs; PRRX2.