Circular RNA circBLNK promotes osteosarcoma progression and inhibits ferroptosis in osteosarcoma cells by sponging miR‑188‑3p and regulating GPX4 expression

As a newly identified circular RNA (circRNA), the role of circBLNK in cancer progression has not been probed. The objective of the present study was to functionally dissect the role of circBLNK in osteosarcoma (OS) tumorigenesis and progression. With regards of the experimental procedure, the levels of mRNAs and proteins were assessed using reverse transcription-quantitative PCR and western blot analysis, respectively. The subcellular location of circBLNK in OS cells was determined by cell cytosolic/nuclear fractionation assay. Cell ferroptosis ability was assessed through MTT assay. Cell proliferative abilities were assessed by clonogenic and Cell Counting Kit-8 assays, and cell apoptosis was measured using flow cytometry. The relationships among circBLNK, miR-188-3p, and glutathione peroxidase 4 (GPX4) were validated by luciferase reporter and RNA pull-down assays, as well as RNA immunoprecipitation. The stability of circBLNK and linear BLNK was confirmed using RNase R treatment assay. The association between circBLNK expression and overall survival rate was assessed by Kaplan-Meier plot. The correlation between the expression levels of circBLNK, miR-188-3p, and GPX4 in OS tissues was assessed by Pearson's χ(2) test. The results revealed that CircBLNK and GPX4 were significantly upregulated in OS tissues, which predicted the poor prognosis. CircBLNK knockdown led to suppressed cell proliferation and enhanced cell apoptosis, an effect that could be reversed by the inhibition of miR-188-3p. In an in vivo circBLNK deficiency model, tumor growth was observed to be markedly suppressed. Moreover, circBLNK deficiency elevated levels of intracellular free iron (Fe(2+)), malondialdehyde, lipid reactive oxygen species and mitochondrial superoxide, while diminishing mitochondrial membrane potential in Erastin-treated OS cells, which were eliminated by overexpressing GPX4. Furthermore, mechanistic investigations revealed that circBLNK sponged miR-188-3p to regulate the expression of GPX4, thereby affecting OS progression. In conclusion, the present study delineated a new regulatory axis involving circBLNK/miR-188-3p/GPX4 in OS progression, adding to the growing evidence that circRNAs are critical gene regulators in cancer progression.