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Circ-HSP90A expedites cell growth, stemness, and immune evasion in non-small cell lung cancer by regulating STAT3 signaling and PD-1/PD-L1 checkpoint

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Abstract

Background

Circular RNAs (circRNAs) are important participators in tumor progression for their stable structure and high tissue-specific expression. The purpose of this research was to clarify the potential and mechanism of a novel circRNA-circ-HSP90A in non-small cell lung cancer (NSCLC).

Methods

Biological potentials of circ-HSP90A in NSCLC were measured by functional assays. Molecular interaction was assessed by bioinformatics analysis and mechanical assays.

Results

Results depicted that circ-HSP90A was cyclization from its host gene heat shock protein 90 alpha (HSP90A) and was up-regulated in NSCLC cells. Circ-HSP90A depletion retarded proliferation, migration, invasion, and immune evasion. Mechanistically, circ-HSP90A recruited ubiquitin specific peptidase 30 (USP30) to stabilize HSP90A and then stimulated the signal transducer and activator of transcription 3 (STAT3) signaling. Meanwhile, circ-HSP90A sponged miR-424-5p to programmed cell death ligand 1 (PD-L1).

Conclusions

Our study firstly showed that circ-HSP90A promoted cell growth, stemness, and immune evasion in NSCLC through regulating STAT3 signaling and programmed cell death 1 (PD-1)/PD-L1 checkpoint, mirroring that targeting circ-HSP90A might become a novel target of immunotherapy in NSCLC.

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Acknowledgements

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Author notes

  1. Jie Lei, Jianfei Zhu and Bengang Hui are co-first authors.

Authors and Affiliations

  1. Department of Thoracic Surgery, The Second Affiliated Hospital of Air Force Medical University, No.569 Xinsi Road, Xi’an, 710038, Shaanxi, China

    Jie Lei, Jianfei Zhu, Bengang Hui, Chenghui Jia, Xiaolong Yan, Tao Jiang & Xiaoping Wang

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Contributions

JL, JZ, and BH contributed to the conceptualization. JL, JZ, BH, CJ, XY, TJ, and XW were involved in the data curation. JL, JZ, BH, CJ, XY, TJ, and XW helped in the formal analysis. TJ and XW were involved in the investigation. TJ and XW contributed to the methodology. JL, JZ, and BH were involved in the project administration. TJ and XW contributed to software. JL, JZ, BH were involved in the supervision. JL, JZ, and Bengang Hui were involved in the validation. JL, JZ, and BH contributed to the visualization. JL, JZ, and BH were involved in writing—original draft preparation. JL, JZ, and BH contributed to writing—review and editing.

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Correspondence to Tao Jiang or Xiaoping Wang.

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Supplementary Information

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262_2022_3235_MOESM1_ESM.tif

Supplementary Figure S1 CircRNAs expression in NSCLC cells. A-O Expression of 15 circRNAs derived from HSP90A was detected in NSCLC cells and BEAS-2B cells via RT-qPCR. *P<0.05, **P<0.01 (TIF 2624 KB)

262_2022_3235_MOESM2_ESM.tif

Supplementary Figure S2 Characterization of circ-HSP90A. A Illustration showed the back-splice junction of circ-HSP90A. B The existence of circ-HSP90A was validated in two NSCLC cells by RT-PCR. Divergent primers amplified circ-HSP90A in cDNA but not in gDNA. GAPDH served as a negative control. C RT-qPCR detected RNA levels of circ-HSP90A and linear-HSP90A from H1299 and A549 cells treated with or without RNase R. D RNA stability of circ-HSP90A and linear-HSP90A in H1299 and A549 cells treated with actinomycin D. **P<0.01 (TIF 1069 KB)

262_2022_3235_MOESM3_ESM.tif

Supplementary Figure S3 HSP90A promotes NSCLC progression. A RT-qPCR and western blot detected the silence efficiency of HSP90A in H1299 and A549 cells. B-C CCK-8 and colony formation experiments examined proliferation in two NSCLC cells upon HSP90A deletion. D-E Wound healing and transwell assays detected migration and invasion in two NSCLC cells upon HSP90A deletion. F Sphere formation assays detected stemness in two NSCLC cells upon HSP90A deletion. G. The expression of HSP90A was detected in H1299 and A549 cells when circ-HSP90A was prohibited. **P<0.01 (TIF 6304 KB)

262_2022_3235_MOESM4_ESM.tif

Supplementary Figure S4 Subcellular localization of circ-HSP90A. A-B Subcellular fractionation and FISH assays determined circ-HSP90A location in two NSCLC cells. C RIP assays detected the enrichment of circ-HSP90A and HSP90A in Ago2. D Luciferase activity of PD-L1 promoter was assessed in two NSCLC cells with circ-HSP90A knockdown. **P<0.01, n.s. meant no significance (TIF 988 KB)

262_2022_3235_MOESM5_ESM.tif

Supplementary Figure S5 Circ-HSP90A promotes the immune evasion of NSCLC via sponging miR-424-5p. A Silence efficiency of miR-424-5p in two NSCLC cells was detected using RT-qPCR. B-C CD8+ T cells were co-cultured with two NSCLC cells, and then transfected with sh-NC, sh-circ-HSP90A#1 and sh-circ-HSP90A#1+miR-424-5p inhibitor. Flow cytometry analysis detected CD8+ T cell percentage and apoptosis. **P<0.01 (TIF 725 KB)

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Lei, J., Zhu, J., Hui, B. et al. Circ-HSP90A expedites cell growth, stemness, and immune evasion in non-small cell lung cancer by regulating STAT3 signaling and PD-1/PD-L1 checkpoint. Cancer Immunol Immunother 72, 101–124 (2023). https://doi.org/10.1007/s00262-022-03235-z

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