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

Abstract

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.

Regulation of ncRNAs involved with ferroptosis in various cancers

“…CircLRFN5 binds to transcription factor PRRX2 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) ( Jiang et al, 2022b ) ( Figure 2M ). Taken together, ncRNAs participate in the regulation of iron transporter SLC40A1 and PRRX2 degradation in GBM.…”

Ferroptosis-associated circular RNAs: Opportunities and challenges in the diagnosis and treatment of cancer

“…CircLRFN5 can mediate paired related homeobox 2 (PRRX2) via the ubiquitin proteasome pathway, and in gliomas, PRRX2 activates transcriptional upregulation of GTP cyclohydrolase 1 (GCH1) expression, a substance that inhibits ferroptosis by producing the antioxidant tetrahydrobiopterin (BH4). This implies that circLRFN5 can mediate ferroptosis in glioma ( Jiang et al, 2022b ).…”

“…CircGFRA1 binding to miR-1228 enhances FSP1 expression to inhibit ferroptosis and promote the development of HER2-positive breast cancer ( Bazhabayi et al, 2021 ). Third, in the GCH1-BH4 pathway, circLRFN5 regulates GCH1-activated ferroptosis via PRRX2 and inhibits glioblastoma progression ( Jiang et al, 2022b ). Moreover, ACSL4 and LPCAT3 mediate the depletion of PUFA-PLs.…”

Synthetic miR-21 decoy circularized by tRNA splicing mechanism inhibited tumorigenesis in glioblastoma in vitro and in vivo models

“… 5 , 6 Circular RNAs (circRNAs) are another class of ncRNAs that are covalently closed and mainly generated through the back-splicing mechanism. 7 circRNAs play essential roles in gene expression and can function as a competitive platform for binding gene regulators such as miRs. circRNAs, by sponging miRs, can act as oncogenes or tumor suppressors in cancer cells.…”