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Mesencephalic astrocyte-derived neurotrophic factor inhibits cervical cancer progression via regulating macrophage phenotype

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Abstract

Background

Cervical cancer is a common gynecologic malignant tumor, but the critical factors affecting cervical cancer progression are still not well demonstrated. Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been widely recognized as an anti-inflammatory factor to regulate macrophage polarization. In this study, the effect and mechanism of MANF on cervical cancer were preliminarily explored.

Methods and results

Kaplan–Meier curve was used to show the overall survival time of the involved cervical cancer patients with high and low MANF expression in cervical cancer tissues. MANF was highly expressed in peritumoral tissues of cervical carcinoma by using immunohistochemistry and western blot. MANF mRNA level was detected by using qRT-PCR. Dual-labeled immunofluorescence showed MANF was mainly expressed in macrophages of cervical peritumoral tissues. Moreover, MANF-silenced macrophages promoted HeLa and SiHa cells survival, migration, invasion and EMT via NF-κB signaling activation. The results of tumor formation in nude mice indicated MANF-silenced macrophages promoted cervical tumor formation in vivo.

Conclusion

Our study reveals an inhibitory role of MANF in cervical cancer progression, indicating MANF as a new and valuable therapeutic target for cervical cancer treatment.

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Data availability

This manuscript includes all data generated and analyzed in this study. More information is available from the corresponding author on request.

Abbreviations

MANF:

Mesencephalic astrocyte-derived neurotrophic factor

HPV:

Human papillomavirus

EMT:

Esenchymal transition

IFN-γ:

Interferon-γ

LPS:

Lipopolysaccharide

IL-4:

Interleukin-4

TNF-α:

Tumor necrosis factor-α

TGF-β:

Transforming growth factor-β

ER:

Endoplasmic reticulum

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Acknowledgements

Not applicable.

Funding

The study was supported by the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (Grant number: 2019PT310002) to Yunxia Cao, and the Research Fund of Anhui Institute of Translational Medicine (Grant number: ZHYX2020A001) to Yunxia Cao.

Author information

Author notes

  1. Miaomiao Huang and Jingjing Hu have contributed equally to this work.

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No. 218 Jixi Road, Hefei, 230022, Anhui, China

    Miaomiao Huang, Jingjing Hu, Yueran Chen & Yunxia Cao

  2. NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), No. 81 Meishan Road, Hefei, 230032, Anhui, China

    Miaomiao Huang, Jingjing Hu & Yunxia Cao

  3. Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People’s Republic of China, No. 81 Meishan Road, Hefei, 230032, Anhui, China

    Miaomiao Huang, Jingjing Hu & Yunxia Cao

  4. Anhui Province Key Laboratory of Reproductive Health and Genetics, No. 81 Meishan Road, Hefei, 230032, Anhui, China

    Miaomiao Huang, Jingjing Hu & Yunxia Cao

  5. Anhui Provincial Engineering Research Center of Biopreservation and Artificial Organs, No. 81 Meishan Road, Hefei, 230032, Anhui, China

    Miaomiao Huang, Jingjing Hu & Yunxia Cao

  6. Anhui Provincial Institute of Translational Medicine, No. 81 Meishan Road, Hefei, 230032, Anhui, China

    Miaomiao Huang, Jingjing Hu & Yunxia Cao

  7. Department of Pathology, The First Affiliated Hospital of Anhui Medical University, No. 218 Jixi Road, Hefei, 230032, Anhui, China

    Yingying Xun

  8. School of Basic Medical Sciences, Anhui Medical University, No. 81 Meishan Road, Hefei, 230032, Anhui, China

    Xinru Zhang

Authors
  1. Miaomiao Huang
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  6. Yunxia Cao
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Contributions

Miaomiao Huang: Conceptualization, Investigation, Supervision, Methodology, Writing-original draft. Jingjing hu: Investigation, Methodology. Yueran Chen: Investigation, Methodology, Formal analysis. Yingying Xun: Methodology. Xinru Zhang: Methodology, Resources. Yunxia Cao: Conceptualization, Investigation, Supervision, Methodology, Funding acquisition.

Corresponding author

Correspondence to Yunxia Cao.

Ethics declarations

Conflict of interest

The authors declare that they have no potential conflicts of interest.

Ethical approval

The Ethical Committee of Anhui Medical University approved this research.

Research involving human and animal rights

All the experiments related to human tissues were conducted in accordance with Helsinki criteria and were performed by the Ethics Committee of First Affiliated Hospital of Anhui Medical University (Approval number: PJ2021-15-28). All patients participating in the study were given informed consent. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health.

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Supplementary Information

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11033_2024_9602_MOESM1_ESM.pdf

Supplementary file1 Figure S1. MANF knockdown in THP-1 cells promoted pro-inflammatory cytokines production and secretion. THP-1 cells were treated with 100 ng/mL phorbol myristate acetate (PMA) for 48 hours following LPS (100 ng/ml) and IFN-γ (20 ng/ml) stimulation for 24 hours to induce M1 macrophage polarization. (A) The protein and (B) mRNA levels of IL-1β, IL-6, CXCL10 and TNF-α were detected by using western blot and qRT-PCR respectively. GAPDH serves as control for normalization. The relative protein levels were calculated. (C) The medium IL-1β, IL-6, CXCL10 and TNF-α levels from THP-1-derived macrophages were detected by using ELISA. THP-1 cells were transfected with siRNA-Con and siRNA-MANF following LPS (100 ng/ml) and IFN-γ (20 ng/ml) stimulation for 24 hours. (D) MANF protein and (E) mRNA levels in THP-1-derived macrophages were decreased after transfection with siRNA-MANF. (F) The protein and (G) mRNA levels of IL-1β, IL-6, CXCL10 and TNF-α in THP-1-derived macrophages were increased after transfection with siRNA-MANF. GAPDH serves as control for normalization. The relative protein levels were calculated. (H) The medium IL-1β, IL-6, CXCL10 and TNF-α levels from THP-1-derived macrophages after transfection with siRNA-MANF were detected by using ELISA. Data are expressed as mean±SEM. **p<0.01, ***p<0.001, n=6. (PDF 207 kb)

11033_2024_9602_MOESM2_ESM.pdf

Supplementary file2 Figure S2. MANF affected EMT and relevant transcription factors of HeLa and SiHa cells. (A) HeLa and SiHa cells were co-cultured in an indirect contact with Control or MANF-silenced macrophages in serum-free medium. The protein levels of Slug, ZEB1 and Twist in HeLa and SiHa cells were detected by using western blot. (B) 5 μg/mL recombinant human MANF protein was added to treat HeLa and SiHa cells. The E-Cadherin and Vimentin protein levels were detected by using western blot. GAPDH serves as control for normalization. (PDF 83 kb)

11033_2024_9602_MOESM3_ESM.pdf

Supplementary file3 Figure S3. MANF knockdown exerted no effect on macrophage proliferation. PCNA level was examined by using western blot in Control or MANF-silenced macrophages. GAPDH serves as control for normalization. (PDF 43 kb)

Supplementary file4 (PDF 3995 kb)

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Huang, M., Hu, J., Chen, Y. et al. Mesencephalic astrocyte-derived neurotrophic factor inhibits cervical cancer progression via regulating macrophage phenotype. Mol Biol Rep 51, 654 (2024). https://doi.org/10.1007/s11033-024-09602-6

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