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Circ_0082374 Promotes the Tumorigenesis and Suppresses Ferroptosis in Non-small Cell Lung Cancer by Up-Regulating GPX4 Through Sequestering miR-491-5p

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Abstract

Circular RNAs (circRNAs) have been identified to be dysregulated in non-small cell lung cancer (NSCLC) and implicated in the progression of this cancer. Here, this work aimed to investigate the role and mechanism of circ_0082374 on NSCLC progression. Levels of circ_0082374, miR-491-5p, GPX4 (glutathione peroxidase 4) and epithelial-mesenchymal transition (EMT)-related proteins were examined by quantitative real-time PCR or western blotting, respectively. Cell proliferation and metastasis were detected using cell counting kit-8, colony formation, EdU, transwell, and Scratch assays. Cell ferroptosis was evaluated by measuring cell survival after the treatment of different ferroptosis inducers or inhibitors, as well as the accumulation of intracellular reactive oxygen species (ROS), ferrous iron (Fe2+) and malondialdehyde (MDA). The binding between miR-491-5p and circ_0082374 or GPX4 was confirmed using dual-luciferase reporter and RNA pull-down assays. In vivo experiments were conducted using murine xenograft assay and immunohistochemistry. Circ_0082374 was a stable circRNA with high expression in NSCLC tissues and cells. Functionally, circ_0082374 silencing suppressed NSCLC cell proliferation and metastasis. Moreover, its down-regulation enhanced ferroptosis by decreasing iron and lipid peroxidation accumulation. Mechanistically, circ_0082374 could indirectly up-regulate GPX4 expression via miR-491-5p, indicating the circ_0082374/miR-491-5p/GPX4 competitive endogenous RNAs (ceRNA) network. Rescue experiments demonstrated that the miR-491-5p/GPX4 axis mediated the regulatory effects of circ_0082374 exerted on NSCLC cells. Moreover, knockdown of circ_0082374 impeded NSCLC growth and EMT via regulating miR-491-5p and GPX4. Circ_0082374 silencing could suppress NSCLC cell proliferation, metastasis and induce ferroptosis through miR-491-5p/GPX4 axis, suggesting a novel therapeutic approach for NSCLC patients.

Graphical Abstract

Circ_0082374 acts as a sponge for miR-491-5p, and up-regulates GPX4 expression through sequestering miR-491-5p, thereby promoting NSCLC cell proliferation, metastasis and suppressing ferroptosis.

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Data Availability

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

Abbreviations

NSCLC:

Non-small cell lung cancer

circRNA:

Circular RNAs

GPX4:

Glutathione peroxidase 4

EMT:

Epithelial-mesenchymal transition

ROS:

Reactive oxyen species

miRNA/miR:

MicroRNA

ceRNA:

Competing endogenous RNA

CCK-8:

Cell counting kit-8

EdU:

5-Ethynyl-2′-deoxyuridine

MDA:

Malondialdehyde

IHC:

Immunohistochemistry

NC:

Negative control

qRT-PCR:

Quantitative real-time PCR

Fe2+:

Ferrous iron

ShRNA:

Short hairpin RNA

Fer-1:

Ferrostatin-1

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Authors and Affiliations

  1. Department of Pulmonary and Critical Care Medicine, Shulan(Hangzhou) Hospital Affiliated to Zhejiang Shuren University Shulan International Medical College, Hangzhou, 310022, Zhejiang, China

    Zongyu Li

  2. Department of General Practice, Shulan(Hangzhou) Hospital Affiliated to Zhejiang, Shuren University Shulan International Medical College, Hangzhou, 310022, Zhejiang, China

    Mengdi Fan

  3. Department of Infectious, Shulan(Hangzhou) Hospital Affiliated to Zhejiang, Shuren University Shulan International Medical College, Hangzhou, 310022, Zhejiang, China

    Zhibo Zhou

  4. Department of Respiratory Therapy, Shulan(Hangzhou) Hospital Affiliated to Zhejiang Shuren University Shulan International Medical College, 848 Dongxin Rd., Hangzhou, 310022, Zhejiang, People’s Republic of China

    Xianyin Sang

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Contributions

Mengdi Fan and Zhibo Zhou designed and performed the research; Xianyin Sang analyzed the data; Zongyu Li wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Xianyin Sang.

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Written informed consents were obtained from all participants and this study was permitted by the Ethics Committee of Shulan (Hangzhou) Hospital Affiliated to Zhejiang Shuren University Shulan International Medical College. The animal study has obtained the assent of the Shulan (Hangzhou) Hospital Affiliated to Zhejiang Shuren University Shulan International Medical College Animal Care and Use Committee.

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Animal studies were performed in compliance with the ARRIVE guidelines and the Basel Declaration. All animals received humane care according to the National Institutes of Health (USA) guidelines.

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12033_2024_1059_MOESM1_ESM.tif

Supplementary file1 (TIF 220 KB)—Circ_0082374/miR-491-5p axis can regulate GPX4 in NSCLC cells. (A, B) The effects of circ_0082374/miR-491-5p axis on GPX4 expression was evaluated by western blotting. **P<0.01, ***P<0.001

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Li, Z., Fan, M., Zhou, Z. et al. Circ_0082374 Promotes the Tumorigenesis and Suppresses Ferroptosis in Non-small Cell Lung Cancer by Up-Regulating GPX4 Through Sequestering miR-491-5p. Mol Biotechnol (2024). https://doi.org/10.1007/s12033-024-01059-z

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