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Synthetic miR-26a mimics delivered by tumor exosomes repress hepatocellular carcinoma through downregulating lymphoid enhancer factor 1

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Abstract

Background

The dysregulation of exosomal microRNAs plays an important role in the progression of hepatocarcinogenesis. In this study, we investigated the therapeutic potential of synthetic exosomal miR-26a against HCC cells and explored the feasibility of tumor-derived exosomes as drug delivery vehicles.

Methods

Proliferation and migration assays were performed to examine the effects of miR-26a on HCC in vitro. The direct target gene of miR-26a was identified through miRecords analysis and target validation. The transferring efficiency and anti-HCC effect of exosomes with different origin were studied and the optimal miR-26a delivery method was established and verified in vitro and in vivo. In addition, the relationships between prognosis of HCC patients and miR-26a expression in HCC serum and exosomes were retrospectively analyzed.

Results

Here, we found that tumor cell-derived exosomes were taken in preferentially by HCC cells and promoted HCC progression through Wnt pathway by low-density lipoprotein receptor-related protein 6 (LRP6). HCC cells with vacuolar protein sorting-associated protein 35 knocked down were adopted to generate engineered LRP6−exosomes. The engineered HCC-derived exosomes loading miR-26a inhibited HCC progression in vitro and in vivo effectively. Overexpression of miR-26a impaired the growth and migration of HCC by targeting lymphoid enhancer factor 1 (LEF1). Moreover, low expression of exosomal miR-26a was an independent prognostic factor for recurrence and survival in HCC patients.

Conclusions

Our findings suggested the exosomal miR-26a could serve as a non-invasive prognostic marker for HCC patients. Genetically modified tumor-derived exosomes showed preferable transfection efficiency but reduced Wnt activity, which provides a novel therapeutic strategy for HCC.

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Availability of data and materials

All data are available in the main text or the supplementary materials. The data used or analyzed during this study are included in this article and available from the corresponding author upon reasonable request.

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Acknowledgements

The authors thank Yong-Zhou Li and Zheng-Tao Zhang for the excellent technical assistance, Qiang Wu, Pei-Yuan Zhang, and Wei Gao for the help with the exosome isolation, identification, and transmission electron microscopy, Yuan Ji and Ling-Li Chen for slide scanning and independent analyses of tissue histopathology, Le Kang and Jing Wang for tissue processing and tail vein injection, Jun Chen, Dong-Mei Gao, and Yan-Yan Nie for mouse model processing help, and Hui-Yan Li and Xiao Guo for the help of IVIS imaging.

Funding

This work was supported by Science and Technology Innovation Plan of Shanghai Science and Technology Commission (NO. 22S11901100; 21Y11912300), Zhongshan Clinical Research Foundation (No. 2020ZSLC48), National Natural Science Foundation of China (Grant No.81401929,81830102, 81772578, and 82002482), Shanghai Rising Star Program (No.16QA1401000), and Natural Science Foundation of Zhejiang Province (No. LQ21H160034).

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Author notes

  1. Jie Hu, Wei-Feng Liu, and Xiang-Yu Zhang have contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, 200032, China

    Jie Hu, Wei-Feng Liu, Xiang-Yu Zhang, Guo-Ming Shi, Xin-Rong Yang, Kai-Qian Zhou, Bo Hu, Fei-Yu Chen, Cheng Zhou, Jia Fan, Zheng Wang & Jian Zhou

  2. Faculty of Medicine, Prince of Wales Hospital, The Chinese University of Hong Kong, Sha Tin, Hong Kong, China

    Wan-Yee Lau

  3. State Key Laboratory of Genetic Engineering and Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Fudan University, Shanghai, 200032, China

    Jia Fan & Jian Zhou

  4. Shanghai Key Laboratory of Organ Transplantation, Zhongshan Hospital, Fudan University, Shanghai, 200032, China

    Jia Fan & Jian Zhou

  5. Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China

    Jia Fan & Jian Zhou

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  1. Jie Hu
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Contributions

JZ, ZW, and JF conceptually designed the experimental strategy, provided intellectual input, and helped write the manuscript. JH, WFL, and XYZ injected mice orthotopically with engineered exosomes, designed the experimental strategy, prepared figures, and wrote the manuscript. KQZ and ZW performed detection of exosomes in tumor tissue sections. XYZ, CZ, XRY, and GMS provided intellectual input, extracted RNA and quantified miRNA loading in exosomes by qPCR analyses, stained tissues for LEF1, and helped with in vivo experiments. XYZ, KQZ, and FYC prepared exosomes (cultured cells for exosome collection and preparation by ultracentrifugation), generated exosomes (electroporation and wash steps involved in the generation of engineered exosome and NanoSight measurements), and treated mice with engineered exosomes. BH provided PDX model and instructed TEXs treatment. WFL, KQZ, and FYC performed the NanoSight measurements, analyses, immunostaining analyses, RNA extraction and qPCR of treated cells, and Western blot analyses, analyzed data, prepared figures, and helped write the manuscript. WFL, JH, WYL, and ZW reviewed the source data, advised on experimental design, and supervised statistical analyses.

Corresponding authors

Correspondence to Zheng Wang or Jian Zhou.

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Conflict of interest

Jie Hu, Wei‑Feng Liu, Xiang‑Yu Zhang, Guo‑Ming Shi, Xin‑Rong Yang, Kai‑Qian Zhou, Bo Hu, Fei‑Yu Chen, Cheng Zhou, Wan‑Yee Lau, Jia Fan, Zheng Wang, Jian Zhou declare that they have no competing interests.

Ethics approval

All blood and tissue samples were collected retrospectively from the specimen bank of Zhongshan Hospital, Fudan University. This study was approved by the Ethics Committee of Zhongshan Hospital (The approval No. B2012-114) and abided the Declaration of Helsinki.

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Hu, J., Liu, WF., Zhang, XY. et al. Synthetic miR-26a mimics delivered by tumor exosomes repress hepatocellular carcinoma through downregulating lymphoid enhancer factor 1. Hepatol Int 17, 1265–1278 (2023). https://doi.org/10.1007/s12072-023-10527-8

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