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CircKLHL2 mitigates septic lung injury via circKLHL2-miR-338-3p-ATF6 ceRNA pathway

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Abstract

Background

Circular RNAs (circRNAs) are newly identified therapeutic targets for sepsis. CircRNA mmu_circ_0001679 dysregulation is underlying the pathogenesis and treatment of sepsis-induced acute lung injury (ALI). Here, the expression and role of its human homologoue circRNA-Kelch-like family member 2 (circKLHL2; hsa_circ_0071374) were explored in lipopolysaccharide (LPS)-induced human ALI.

Objective

Expression levels of circKLHL2, microRNA (miR)-338-3p and activating transcription factor 6 (ATF6) were detected by quantitative PCR and western blotting, and dual-luciferase reporter assay confirmed target relationships among them. Cell injury was measured using cell-counting kit-8, reactive oxygen species (ROS)/malondialdehyde (MDA)/superoxide dismutase (SOD) assays, Annexin V-fluorescein isothiocyanate apoptosis assay, western blotting and cysteine-requiring aspartate proteases (caspases) activity assays.

Result

The expression of circKLHL2 and ATF6 was upregulated in sepsis-damaged human lung tissues and LPS-stressed human bronchial epithelial cells (16HBE), with a concomitant decrease of miR-338-3p. RNA interfering of circKLHL2 improved cell viability of LPS-challenged 16HBE cells, and suppressed apoptosis, oxidative stress and endoplasmic reticulum (ER) stress, as evidenced by the increased bcl-2 level and SOD activity, and the decreased apoptosis rate, caspase3/9 activity and levels of bcl-2-associated X protein, ROS, MDA, ATF6 and C/EBP-homologous protein. Moreover, circKLHL2 knockdown exerted similar results to ER stress inhibitor 4-phenyl butyric acid in LPS injury. In addition, circKLHL2 knockdown reversed LPS-induced mitochondrial dysfunction. ATF6 and circKLHL2 were competing endogenous RNAs of miR-338-3p, and either exhausting miR-338-3p or restoring ATF6 could counteract circKLHL2 knockdown-mediated roles in LPS injuries.

Conclusion

Depleting circKLHL2 suppressed LPS-induced ALI through suppressing mitochondrial dysfunction and cytotoxicity and oxidative and ER stresses-induced apoptosis via circKLHL2-miR-338-3p/ATF6 axis.

Highlights

  1. 1.

    CircKLHL2 was upregulated in sepsis-damaged lung tissues and LPS-induced bronchial epithelial cells.

  2. 2.

    Interfering circKLHL2 attenuated LPS-elicited cytotoxicity, apoptosis, and oxidative and ER stresses, and mitochondrial dysfunction.

  3. 3.

    ATF6 upregulation and miR-338-3p downregulation counteracted the role of circKLHL2 depletion.

  4. 4.

    CircKLHL2 and ATF6 were ceRNAs for miR-338-3p.

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Authors and Affiliations

  1. Department of Critical Medicine, China-Japan Union Hospital of Jilin University, No.126 Xiantai Street, Changchun, 130033, Jilin, China

    Chunmei Zhang, Ruoran Wu & Zhongyan Zhao

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  1. Chunmei Zhang
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Contributions

All authors have been involved in the management of the patient and in the conception of the manuscript. ZCM and ZZY have been involved in the drafting of the manuscript or its critical revision for important intellectual content. WRR and ZZY have been involved in data collection. All authors have read and approved the final manuscript.

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Correspondence to Zhongyan Zhao.

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Author Chunmei Zhang declares that he/she has no conflict of interest, author Ruoran Wu declares that he/she has no conflict of interest, and the author Zhongyan Zhao declares that he/she has no conflict of interest.

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This study protocol was reviewed and approved by China-Japan Union Hospital of Jilin University and written informed consents were obtained from all participants.

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13273_2023_349_MOESM1_ESM.tif

Supplementary file1 Role of ER stress inhibitor in LPS-stimulated 16HBE cells. (A-H) 10 μg/mL of LPS-administrated 16HBE cells were pre-treated with 5 mM 4-PBA for 2 h. (A) CCK8 assay measured the percentage of cell viability. (B) Annexin V-FITC/PI staining and FCM determined the percentage of apoptotic cells. (C) Caspase3 and caspase9 activity assays tested the indicated activity. (D, H) Western blotting detected protein expression of bcl-2, bax, ATF6, CHOP, and GAPDH (as loading control). (E-G) ROS, MDA and SOD assay kits tested ROS level, MDA level and SOD activity, respectively. *P<0.05. (TIF 1537 KB)

13273_2023_349_MOESM2_ESM.tif

Supplementary file2 Role of circKLHL2 exhaustion in cytotoxicity, apoptosis, and oxidative and ER stresses. (A-I) 16HBE cells were expressed with sh-NC and sh-circKLHL2 via 24 h-transfection, and then insulted by 10 μg/mL of LPS for 24 h. (A) QPCR examined circKLHL2 expression. (B) CCK8 assay measured the percentage of cell viability. (C) Annexin V-FITC/PI staining and FCM determined the percentage of apoptotic cells. (D) Caspase3 and caspase9 activity assays tested the indicated activity. (E, I) Western blotting detected protein expression of bcl-2, bax, ATF6, CHOP, and GAPDH (as loading control). (F-H) ROS, MDA and SOD assay kits tested ROS level, MDA level and SOD activity, respectively. *P<0.05. (TIF 1183 KB)

13273_2023_349_MOESM3_ESM.tif

Supplementary file3 Role of circKLHL2/miR-338-3p/ATF6 axis in mitochondrial dysfunction. (A) Evaluation of ΔΨm in 16HBE cells by FCM. (B) Detection of ATP levels in 16HBE cells. (C) Relative mtDNA copy numbers in 16HBE cells. *P<0.05. (TIF 338 KB)

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Zhang, C., Wu, R. & Zhao, Z. CircKLHL2 mitigates septic lung injury via circKLHL2-miR-338-3p-ATF6 ceRNA pathway. Mol. Cell. Toxicol. 20, 353–365 (2024). https://doi.org/10.1007/s13273-023-00349-y

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